WO2024186890A1 - Compositions and methods for hepatitis b virus (hbv) genome editing - Google Patents

Compositions and methods for hepatitis b virus (hbv) genome editing Download PDF

Info

Publication number
WO2024186890A1
WO2024186890A1 PCT/US2024/018654 US2024018654W WO2024186890A1 WO 2024186890 A1 WO2024186890 A1 WO 2024186890A1 US 2024018654 W US2024018654 W US 2024018654W WO 2024186890 A1 WO2024186890 A1 WO 2024186890A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotides
sequence
seq
optionally
hbv
Prior art date
Application number
PCT/US2024/018654
Other languages
French (fr)
Inventor
William Frederick HARRINGTON
Aishwarya Prakash JAGTAP
Original Assignee
Intellia Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Intellia Therapeutics, Inc. filed Critical Intellia Therapeutics, Inc.
Publication of WO2024186890A1 publication Critical patent/WO2024186890A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • Hepatitis B virus is an enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation, and is a major risk factor for development of cirrhosis and hepatocellular carcinoma (HCC).
  • WHO World Health Organization
  • a number of therapeutic agents have been developed for the treatment of HBV that effectively reduce the disease burden of HBV infection, but they are not typically curative as they do not fully eliminate all replicative forms of the virus including the covalently closed circular DNA (cccDNA) that resides in the hepatocyte nucleus and becomes a template for viral replication and transcription of viral RNAs.
  • Nucleotide and nucleoside analogs typically considered to be the gold standard for treatment of chronic HBV infection due to their safety and efficacy, effectively suppress HBV replication, but do not eliminate cccDNA, do not prevent expression of viral proteins, must be dosed chronically, and can result in the development of resistance.
  • HCC hepatocellular carcinoma
  • Interferon-based therapies can result in seroconversion and cure about 10-15% of patients, thereby allowing discontinuation of treatment, but the agents have severe side effects and must be refrigerated for long-term storage, making them less desirable for use in many countries where HBV infection is prevalent.
  • Treatment of chronic HBV infection is further complicated by the ability of HBV to evade or suppress the immune response resulting in persistence of the infection.
  • HBV proteins have immune-inhibitory properties, with hepatitis B s antigen (HBsAg) comprising the overwhelming majority of HBV protein in the circulation of HBV-infected subjects. Additionally, while the removal (via seroconversion) of hepatitis B e antigen (HBeAg) or reductions in serum viremia are not correlated with the development of sustained control of HBV infection off treatment, the removal of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-recognized prognostic indicator of antiviral response to treatment that will lead to control of HBV infection off treatment. However, this only occurs in a small fraction of patients receiving immunotherapy.
  • Hepatitis D virus or hepatitis delta virus is a human pathogen.
  • the virus is defective and depends on obligatory helper functions provided by the hepatitis B virus (HBV) for transmission; indeed, HDV requires a simultaneous infection with HBV (co- infection) or superimposition on a pre-existing HBV infection (superinfection) to become infectious and thrive.
  • HBV hepatitis B virus
  • HDV requires the HBV viral envelope containing the surface antigen of hepatitis B. Therefore, an effective treatment of HBV infection would provide an effective treatment of HDV.
  • compositions and methods for modifying the hepatitis B virus (HBV) genome are also provided for treating a subject infected with HBV.
  • a guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 conitguous nucleotides of nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; B.
  • a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; C.
  • a targeting sequence comprising a sequence identical to or complementary to at least 17, 18, preferably 19, or 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1; or D.
  • a targeting sequence comprising a sequence identical to or complementary to at least 20, 21, 22, preferably 23, or 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1.
  • a guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of: 1.
  • nucleotides 163-466 nucleotides 1- 843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821- 2460 of SEQ ID NO: 1; 2.
  • a target site selected from a target site of any one of HBV number S1-S93 in Table 1A optionally a target site of any one of HBV number S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 in Table 1A; B.
  • a targeting sequence comprising a sequence identical to or complementary to 17, 18, preferably 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, or to a targeting sequence of S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 provided in Table 1A; C.
  • a targeting sequence comprising a sequence identical to or complementary to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1- 1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460; or nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250- 269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686- 705, 694-716, 694-713, 695-714, 6
  • the guide RNAs provided herein further comprise one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1- 8, b.
  • the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 268); or 3.
  • the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or C.
  • the guide RNA lacks 6 nucleotides in shortened hairpin 1.
  • the guide RNA lacks 8 nucleotides in shortened hairpin 1. [013] In some embodiments, H-1 and H-3 are deleted. [014] In some embodiments, the guide RNA further comprises a 3’ tail. [015] In some embodiments, the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length. [016] In some embodiments, the guide RNA comprises an upper stem region comprising a modification to any one or more of US1-US12 in the upper stem region. [017] In some embodiments, the guide RNAs described herein comprise a nucleotide sequence selected from the sequences in Table 3A.
  • the guide RNA comprises a modified nucleotide sequence selected from the modified Spy guide scaffold sequences in Table 4, wherein the modified nucleotide sequence is 3’ of the guide sequence.
  • the guide RNAs described herein are modified according to the pattern of a nucleotide sequence selected from the modified Spy guide RNA sequences in Table 15.
  • the guide comprises a nucleotide sequence selected from the unmodified Spy guide RNA Sequences in Table 3B, wherein the N20’s are collectively a targeting sequence described herein.
  • each nucleotide of the unmodified Spy guide RNA Sequences in Table 3B is any natural or non-natural nucleotide.
  • the guide RNA is modified according to a pattern selected from the modification patterns in Table 15, wherein the (mN*)3N17 refers to the targeting sequence described herein in which the first three nucleotides comprises a 2’-O-Me modification and a phosphorothioate linkage.
  • the guide RNAs described herein comprise a sequence or modification pattern selected from SEQ ID NOs: 269, 273, 274, 362, 366, or 367 as set forth in Table 12.
  • a guide RNA comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of: 1. nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; 2.
  • a target site selected from any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; B.
  • the guide region comprises a nucleic acid sequence identical to or complementary to 21, 22, preferably 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; or of a targeting sequence of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; C.
  • the guide region is selected from targeting sequence of G027224, G027225, G027228, G030107, G030108, G030115, G030120, G030123, G030125, G030130, G030134, G030138, G030158, G030162, G030167, G030180, or G030181; optionally G027224, G027225, G027228, G030107, G030115, G030120, G030123, G030125, G030130, G030162, or G030167; optionally G027224, G030107, G030115, or G030120; or D.
  • the guide region comprising a sequence identical to or complementary to 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1; or nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740- 763, 755-778, 1390-1413, 2951-2974, or 29
  • the conserved region comprises one or more of: (a) a shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region lacks 2-24 nucleotides relative to SEQ ID NO: 455, wherein (i) one or more of nucleotides 37-48 and 53-64 is deleted relative to SEQ ID NO: 455 and optionally one or more of nucleotides 37-64 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2- 10 nucleotides, optionally 2-8 nucleotides relative to SEQ ID NO: 455 wherein (i) one or more of nucleotides 82-86 and 91-95 is deleted relative to SEQ ID NO: 455 and optionally one or more of positions 82-96 is substituted relative
  • the conserved region comprises a nucleotide sequence selected from Table 5.
  • the guide RNA comprises at least one end modification.
  • the modification comprises a 5’ end modification.
  • the modification comprises a 3’ end modification.
  • the guide RNA comprises a modification in a hairpin region.
  • the modification in a hairpin region is also an end modification.
  • the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide.
  • the modification comprises a phosphorothioate (PS) bond between nucleotides.
  • the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide.
  • the modification comprises a 2’-fluor (2’F) modified nucleotide.
  • the 5’ end modification comprises a 2’-O-methyl (2’-O- Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide at nucleotides 1-3 of the 5’ end of the guide sequence.
  • the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motifs in Table 6, and wherein the conserved region is 3’ of the guide region.
  • the guide RNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 226-234, and wherein the N’s represent the guide sequence of any one of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37.
  • each nucleotide is any natural or non-natural nucleotide.
  • the guide RNA is modified according to a pattern selected from SEQ ID NOs: 670-678, wherein the N’s are collectively the guide sequence described herein, wherein N, A, C, G, and U are ribonucleotides (2’-OH), wherein “m” indicates a 2’-O- Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides.
  • a composition comprising a guide RNA described herein.
  • composition comprising a combination of guide RNAs comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of target sites selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N37; N24 and N79; N24, N32, and N79; N24, N32, and N37; and N24, N32, and N40; optionally N24 and N37; N7 and N32; N7 and N37; and B.
  • the conserved region comprises a conserved region described herein.
  • the composition further comprises an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
  • the nucleic acid encoding the RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding the RNA-guided DNA binding agent.
  • the RNA-guided DNA binding agent is a nuclease .
  • the RNA-guided DNA binding agent is a Cas9 nuclease. [047] In some embodiments, the Cas9 is S.
  • the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 707 and 713 or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to a sequence selected from SEQ ID NOs: 707 and 713.
  • the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706 and 712.
  • the Cas9 is Nme Cas9.
  • Nme Cas9 comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 711, 716, 719, 722, or 730 or an ORF encoding an Nme Cas9 having at least 90% identity to a sequence selected from 600-620.
  • the ORF encoding the amino acid sequence has at least 85% identity to a sequence selected from SEQ ID NOs: 705, 710, 715, 718, 721, or 729.
  • the nuclease has double stranded endonuclease activity.
  • the nuclease has nickase activity.
  • the nuclease is catalytically inactive.
  • the nuclease further comprises a heterologous functional domain.
  • the nuclease is a nickase and the heterologous functional domain is a deaminase.
  • the deaminase is a cytidine deaminase or an adenine deaminase.
  • the deaminase is a cytidine deaminase.
  • the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.
  • APOBEC apolipoprotein B mRNA editing enzyme
  • the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs.707, 716, 719, 722, or 730 or an ORF encoding an amino acid sequence having at least 90% identity to SEQ ID NOs: 707, 716, 719, 722, or 730.
  • the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706, 715, 718, 721, or 729.
  • the composition described herein further comprises a uracil glycosylase inhibitor (UGI) or nucleic acid encoding a UGI, wherein the nuclease polypeptide does not comprise a UGI or the nucleic acid encoding the polypeptide does not encode a UGI.
  • UGI comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 709, 725, or 733 or an ORF encoding an amino acid sequence having at least 90% identity to a sequence selected from 709, 725, or 733.
  • the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NO: 708, 724, 732, optionally SEQ ID NO: 708.
  • the ORF is a modified ORF.
  • the composition described herein further comprises a pharmaceutical excipient.
  • the guide RNA is associated with a lipid nanoparticle (LNP).
  • the LNP comprises a cationic lipid.
  • the cationic lipid is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.
  • the LNP comprises a helper lipid.
  • the helper lipid is cholesterol.
  • the LNP comprises a neutral lipid.
  • the neutral lipid is 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).
  • the LNP comprises a stealth lipid.
  • the stealth lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol 2000 (PEG2k-DMG).
  • the LNP comprises (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.
  • a pharmaceutical composition comprising the guide RNA described herein or the composition described herein.
  • a pharmaceutical composition comprising, or use of, the guide RNA described herein or the composition described herein for inducing a double stranded break or a single stranded break within an HBV gene in a cell, modifying the nucleic acid sequence of an HBV gene in a cell, or reducing expression of an HBV gene in a cell.
  • the HBV gene is the S gene.
  • the pharmaceutical composition or use described herein is for reducing expression of the HBV gene in a cell or subject.
  • a pharmaceutical composition comprising, or use of, the guide RNA described herein or the composition described herein for treating a subject having hepatitis B.
  • the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the pharmaceutical composition.
  • the pharmaceutical composition or use described herein is for use in combination with an immune stimulator.
  • the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha-2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1
  • the immune stimulator is a vaccine.
  • the vaccine is a nucleic acid vaccine or a protein-based vaccine.
  • the nucleic acid vaccine is an RNA vaccine.
  • the subject has chronic HBV.
  • the subject is HBe antigen positive.
  • the subject is HBe antigen negative.
  • the subject has hepatitis D.
  • the pharmaceutical composition or use described herein is for treating hepatitis D.
  • a method or inducing a double stranded break or a single stranded break within an HBV gene in a cell or reducing expression of an HBV protein in a cell comprising contacting the cell with the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein.
  • the HBV protein is HBsAg protein or HBcrAg protein.
  • the cell is in a subject.
  • the HBsAg protein level or the HBcrAg protein level is measured in a subject sample selected from blood or serum.
  • the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml.
  • the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition.
  • the method described herein further comprise administration of an immune stimulator.
  • the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha-2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1
  • the immune stimulator is a vaccine.
  • the vaccine is a nucleic acid vaccine or a protein-based vaccine.
  • the nucleic acid vaccine is an RNA vaccine.
  • the subject has chronic HBV.
  • the subject is HBe antigen positive.
  • the subject is HBe antigen negative.
  • the subject has hepatitis D.
  • the methods described herein further comprise treating hepatitis D in the subject.
  • the method comprises administering one or more doses of the guide RNA and the RNA-guided DNA binding agent, of the nucleic acid encoding an RNA- guided DNA binding agent, or of the composition to a subject, optionally comprising administering two, three, four, or five doses.
  • the method described herein comprises administering two or more of the doses, wherein at least two consecutive doses are administered at times separated by at least or about 1 month, 3 months, or 6 months.
  • the method described herein comprises, after administering a first dose, determining HBsAg protein level or HBcrAg protein level in blood or serum of the subject; and subsequently administering to the subject a second dose if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml.
  • the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose.
  • a liver cell comprising an HBV genome, wherein the HBV genome comprises a sequence having at least 80%, 85%, optionally 90% or 95% identity, optionally 100% identity to at least 20 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356- 375, 361-380, 364-3
  • the genetic modification comprises a mutation that generates a stop codon or disrupts initiation of transcription.
  • the genetic modification comprises an indel.
  • the HBV genome comprises a sequence having at least 90%, 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356- 375, 361-380, 364-383, 373-392, 423-445, 423-442,
  • the liver cell comprises a second genetic modification in the HBV genome within the sequence having at least 80%, 85%, optionally 90% or 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-6
  • the genetic modification is within the HBV genome within the sequence having at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity identity to at least 20 contiguous nucleotides of targeting sites in Table 1A, optionally a targeting site of a gRNA with an HBV number selected from S1-S6, S8, S11, S16, S17, S19, S21-25, S28- S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80- S87, S80, S90-S91, and S93, optionally S1, S5, or S6.
  • the HBV genome comprises a sequence having 100% identity or at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-7
  • the HBV genome comprises a sequence having at least 90% or 95% identity; or 100% identity to at least 24 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846 , or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778
  • the genetic modification is within the sequence having at least 80%, 85%, optionally 90%, or 95% identity or 100% identity to at least 24 contiguous nucleotides of the target sites in Table 2A, optionally a targeting site of a gRNA with an HBV number selected from N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37; or a targeting site of gRNAs with a combination of HBV numbers selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N
  • the HBV genome is integrated into the liver cell genome. [0125] In some embodiments, the HBV genome is cccDNA. [0126] In some aspects, provided herein a method of modifying a nucleic acid sequence of the HBV genome, the method comprising contacting an HBV-infected liver cell the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein. [0127] In some embodiments, the method is performed in vivo, in vitro, or ex vivo. [0128] In some embodiments, the cell is in a subject infected with HBV.
  • a method of treating an HBV infection in a subject comprising administering to the subject the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein.
  • the nucleic acid sequence of the HBV genome encodes an HBV protein.
  • the HBV protein is HBsAG or ABcrAg protein.
  • the method further comprises determining the HBsAg protein level or the HBcrAg protein level in a subject blood or serum sample.
  • the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml.
  • the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition.
  • the methods described herein further comprise administering an immune stimulator to the subject. DETAILED DESCRIPTION [0136]
  • nucleotide base pairs As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified. [0145] As used herein, ranges include both the upper and lower limit. [0146] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.
  • detecting an analyte and the like is understood as performing an assay in which the analyte can be detected, if present, wherein the analyte is present in an amount above the level of detection of the assay.
  • 100% inhibition is understood as inhibition to a level below the level of detection of the assay
  • 100% encapsulation is understood as no material intended for encapsulation can be detected outside the vesicles.
  • a nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
  • Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy, 2’ halide, or a 2’-O-(2-methoxyethyl) (2’-O-moe) substitutions.
  • RNA may comprise one or more deoxyribose nucleotides, e.g. as modifications, and similarly a DNA may comprise one or more ribonucleotides.
  • Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1- methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-di
  • Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No.5,585,481).
  • a nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional nucleosides with 2’ methoxy substituents, or polymers containing both conventional nucleosides and one or more nucleoside analogs).
  • Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41).
  • Nucleic acid includes “unlocked nucleic acid” enables the modulation of the thermodynamic stability and also provide nuclease stability.
  • RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.
  • Polypeptide refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like. [0153] “Guide RNA,” “gRNA,” and simply “guide” are used herein interchangeably to refer to, for example, either a single guide RNA or the combination of a crRNA and a trRNA (also known as tracrRNA).
  • the crRNA and trRNA may be associated as a single RNA strand (as a single guide RNA, sgRNA) or, for example, in two separate RNA strands (dual guide RNA, dgRNA).
  • Guide RNA or “gRNA” refers to each type.
  • the trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations.
  • a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent.
  • a “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.”
  • a guide sequence can be about 20 nucleotides in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9), or guide sequence can be about 24 nucleotides in length, e.g., in the case of Neisseria meningitidis (Nme Cas9), and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used.
  • Spy Cas9 guides can be 16-, 17-, preferably 18-, 19-, or 20- nucleotides in length, such that, in some embodiments, the Spy Cas9 targeting sequence comprises 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, from nucleotides 153- 173, 245-269, 153-172, 154-173, 245-264, 248-267, 250-269, 364-383, 621-665, 694-716, 1176- 1216, 1187-1216, 1383-1418, 1398-1418, 1424-1448, 1701-1724, 2296-2318, 2867-2894, 2901- 2928, 2976-3001 , 621-640, 629-648, 636-655, 694-713, 695-714, 697-716, 796-815, 11
  • Spy Cas9 guides can be 16-, 17-, preferably 18-, 19-, or 20- nucleotides in length, such that, in some embodiments, the Spy Cas9 targeting sequence comprises 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250- 269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686- 705, 694-716, 694-713, 695-714, 697-716, 713-732
  • Table 1A HBV Spy-Cas9 guide sequences and sequence coordinates [0158]
  • Table 1B the guide targeting sequences for gRNA to be used with a Spy-Cas9 show significant cross-reactity to different strains of HBV.
  • a single gRNA may be suitable for Spy-Cas9-mediated editing of the genomes of more than one HBV strains.
  • multiple gRNAs can be administered to increase the likelihood that the unknown HBV strain genome will be edited.
  • the targeting sequence is in an open reading frame or in a genome, e.g., a viral genome, for example, and is complementary to the guide sequence.
  • the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%, or is 100%.
  • the targeting sequence comprises a sequence that is at least 80%, 85%, preferably 90%, or 95%, or is 100% identical or complementary to 20 contiguous nucleotides of a sequence selected from nucleotides 163- 466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 57-76, 66- 85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713,
  • the targeting sequence and the target region may be 100% complementary or identical to at least 20 contiguous nucleotides of nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364- 383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697- 716, 713-732, 725-744, 1597-1616, 1817
  • the guide sequence and the target region may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence.
  • the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches within the duplex formed between the guide and the target sequence, where the total length of the target sequence is 16, 17, 18, 19, 20 nucleotides, or more.
  • the guide sequence and the target sequence may contain 1-4 mismatches where the guide sequence comprises at least 20 nucleotides.
  • the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.
  • the guide sequence and the target region may form a duplex region having 16, 17, 18, 19, 20 base pairs, or more.
  • the duplex region may include 1, 2, 3, or 4 mismatches such that guide strand and target sequence are not fully complementary.
  • a guide strand and target sequence may be complementary over a 20 nucleotide region, including 2 mismatches, such that the guide sequence and target sequence are 90% complementary providing a duplex region of 18 base pairs out of 20.
  • More tolerated mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches.
  • Nme guide targeting sequences can be 19, 20, 21, preferably 22, 23, or 24 nucleotides in length such that, in some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447- 470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176,
  • Table 2A HBV Nme-Cas9 guide sequences and sequence coordinates [0161]
  • Table 2B the guide targeting sequences for gRNA to be used with an Nme-Cas9 show significant crossreactity to different strains of HBV.
  • a single gRNA may be suitable for Nme-Cas9-mediated editing of the genomes of more than one HBV strains.
  • multiple gRNAs can be administered to increase the likelihood that the unknown HBV strain genome will be edited.
  • the target sequence is in an open reading frame or a in a genome, e.g., a viral genome, and is complementary to the targeting sequence.
  • the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%.
  • the guide sequence comprises a sequence 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 35-58, 146-169, 153-176, 216-239, 330- 356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 4
  • the targeting sequence and the target sequence may be 100% complementary or identical.
  • the targeting sequence and the target sequence may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence.
  • the targeting sequence and the target sequence may contain 1-2, preferably no more than 1 mismatch, where the total length of the target sequence is 19, 20, 21, 22, preferably 23, or 24, nucleotides, or more.
  • the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises at least 24 nucleotides.
  • the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises 24 nucleotides.
  • the targeting sequence and the target sequence may form a duplex region having 24 base pairs, or more.
  • the duplex region may include 1-2 mismatches such that targeting sequenceand target sequence are not fully complementary. Mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Edraki et al., 2019. Mol. Cell, 73:1-13). [0163] Target sequences for RNA-guided DNA binding agents, as defined by the targeting sequence of a guide RNA, may be present on either the positive or negative strand.
  • Tables and other disclosures provided herein may recite genomic coordinates or position within a nucleotide sequence as a target sequence.
  • the guide can be complementary to either the positive or negative strand of the DNA as defined by the genomic coordinates or position within a nucleotide sequence.
  • the sequence to which the guide is complementary depends on the presence of an appropriate PAM for the RNA guided DNA binding protein on the opposite strand.
  • the targeting sequence binds the reverse complement of a target sequence, i.e., the targeting sequence is identical to certain nucleotides of the sense (positive) strand of the target sequence, when the PAM is present in the sense strand, except for the substitution of U for T in the targeting sequence.
  • RNA-guided DNA binding agent means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the presence of a PAM and the sequence of the guide RNA.
  • exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”).
  • Cas nickases include nucleases in which one of the RuvC or HNH domain of the Cas protein, such that only a single strand is cleaved by the nuclease
  • the dCas DNA binding agent may be a dead nuclease comprising non-functional nuclease domains (RuvC or HNH domain).
  • the Cas cleavase or Cas nickase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g., via fusion with a FokI domain.
  • Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.
  • a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity.
  • Class 2 Cas nucleases include Class 2 Cas cleavases/nickases (e.g., H840A, or D10A variants of Spy Cas9 and D16A and H588A of Nme Cas9, e.g., Nme2 Cas9), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated.
  • Class 2 Cas cleavases/nickases e.g., H840A, or D10A variants of Spy Cas9 and D16A and H588A of Nme Cas9, e.g., Nme2 Cas9
  • Class 2 dCas DNA binding agents in which cleavase/nickase activity is inactivated.
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof.
  • Cas9 Cas9
  • Cpf1, C2c1, C2c2, C2c3, HF Cas9 e.g., N497A, R661A, Q695A, Q926A variants
  • HypaCas9 e.g., N692A, M694
  • Cpf1 protein Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain.
  • Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).
  • Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided below.
  • nucleotide sequence encoding Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Sequences with at least 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 amino acid sequences provided herein are also contemplated.
  • the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.
  • Exemplary open reading frames and amino acid sequences for Cas9 (SEQ ID NO: 705, 706, 707, 710, 711, 712, 713, 715, 716, 718, 719, 721, 722, 729, 730) and uracil glycosylase inhibitors (SEQ ID NO: 708, 709, 724, 725, 732, 733) are provided in Table 40.
  • a “cytidine deaminase” means a polypeptide or complex of polypeptides that is capable of cytidine deaminase activity, that is catalyzing the hydrolytic deamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine.
  • Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol.22:367-77, 2005; Conticello, Genome Biol.9:229, 2008; Muramatsu et al., J. Biol.
  • variants of any known cytidine deaminase or APOBEC protein are encompassed.
  • Variants include proteins having a sequence that differs from wild-type protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions.
  • a shortened sequence could be used, e.g., by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence.
  • the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to a reference sequence.
  • the variant is “functional” in that it shows a catalytic activity of DNA editing.
  • the term “APOBEC3A” refers to a cytidine deaminase such as the protein expressed by the human A3A gene.
  • the APOBEC3A may have catalytic DNA editing activity.
  • An amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 734.
  • the APOBEC3A protein is a human APOBEC3A protein or a wild-type protein.
  • Variants include proteins having a sequence that differs from wild-type APOBEC3A protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions.
  • a shortened APOBEC3A sequence could be used, e.g. by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C- terminus of the sequence.
  • an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence).
  • an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).
  • Nme2Cas9 meningitidis (Esvelt et al., Nat. Meth., vol.10, 2013, 1116 - 1121; Hou et al., PNAS, vol.110, 2013, pages 15644 - 15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9).
  • the Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes an N4CC PAM, and can be used for in vivo editing (Ran et al., Nature, vol.520, 2015, pages 186 - 191; Kim et al., Nat. Commun., vol.8, 2017, pages 14500).
  • Nme2Cas9 has been shown to be naturally resistant to off-target editing (Lee et al., Mol. Ther., vol.24, 2016, pages 645 - 654; Kim et al., 2017). See also e.g., WO/2020081568 (e.g., pages 28 and 42), describing an Nme2Cas9 D16A nickase, the contents of which are hereby incorporated by reference in its entirety.
  • “NmeCas9” or “Nme Cas9” is generic and an encompasses any type of NmeCas9, including, Nme1Cas9, Nme2Cas9, and Nme3Cas9.
  • fusion protein refers to a hybrid polypeptide which comprises polypeptides from at least two different proteins or sources.
  • One polypeptide may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy- terminal (C- terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively.
  • Any of the proteins provided herein may be produced by any method known in the art.
  • the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker.
  • uracil glycosylase inhibitor refers to a protein that is capable of inhibiting a uracil-DNA glycosylase (UDG) base-excision repair enzyme (e.g., UniPROT ID: P14739; SEQ ID NO: 735).
  • linker refers to a chemical group or a molecule linking two adjacent molecules or moieties. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). Exemplary peptide linkers are disclosed elsewhere herein. Exemplarly linkers for use in gRNAs are disclosed elsewhere herein.
  • Modified uridine is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine.
  • a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton.
  • a modified uridine is pseudouridine.
  • a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton.
  • a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine, e.g., N1-methyl-psuedouridine.
  • “Uridine position” as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine.
  • a polynucleotide in which “100% of the uridine positions are modified uridines” contains a modified uridine at every position that would be a uridine in a conventional RNA (where all bases are standard A, U, C, or G bases) of the same sequence.
  • a U in a polynucleotide sequence of a sequence table or sequence listing in or accompanying this disclosure can be a uridine or a modified uridine.
  • ribonucleoprotein or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9).
  • the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with the target sequence and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.
  • RNA-guided DNA binding agent means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA.
  • exemplary RNA-guided DNA binding agents include Cas cleavases (which have double strand cleaving activity), Cas nickases (which have single strand cleaving activity), and inactivated forms thereof (“dCas DNA binding agents”).
  • Cas nuclease encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents.
  • the dCas DNA binding agent may be a dead nuclease comprising non-functional nuclease domains (RuvC or HNH domain).
  • the Cas cleavase or Cas nickase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g., via fusion with a FokI domain.
  • the RNA-guided DNA binding agent has nuclease activity, e.g., cleavase or nickase activity.
  • a “control” is understood as an appropriate matched sample or subject for comparison.
  • a control can be a cell population treated in the same manner as the test population except that the treatment used for the control population lacks at least one active agent, e.g., a guide RNA, an mRNA encoding a nuclease, an insertion construct, a lipid formulation.
  • a control may be an internal control, e.g., a cell population or subject prior to treatment.
  • a “control” as in a control subject is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease.
  • a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention.
  • a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treated subject, to provide a normal control, e.g., an enzyme concentration or activity in a subject sample.
  • a normal control can be a population control, i.e., the average of subjects in the general population.
  • a control can be an untreated subject with the same disease.
  • a control can be a subject treated with a different therapy, e.g., the standard of care.
  • a control can be a subject or a population of subjects from a natural history study of subjects with the disease of the subject being compared. In certain embodiments, the control is matched for certain factors to the subject being tested, e.g., age, gender. In certain embodiments, a control may be a control level for a particular lab, e.g., a clinical lab. The ability to design or select appropriate controls is within the ability of those of skill in the art. It is understood when relative values are provided, they can be considered as relative values as compared to an appropriate control.
  • purified such as in “purified protein” or “purified nucleic acid” is understood as being substantially removed from the mixture in which it was made, e.g., a cell, a subject sample, a reaction mixture. In certain embodiments, purified is understood as at least 50% of the mixture is the purified compound, e.g., purified protein or purified nucleic acid by weight. In certain embodiments, purified is understood as at least 80% of the mixture is the purified compound by weight.
  • subject includes primates, including human and non-human primates, mouse, and rat. In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a non-human subject.
  • the subject is a non-human subject expressing one or more human genes, e.g., a transgenic mouse expressing a human gene, a mouse in which the liver has been repopulated with human hepatocytes (e.g., PXB-mouse®).
  • a “target sequence” refers to a sequence of nucleic acid in a target genome or open reading frame, in either the positive or the negative strand, that has complementarity to the guide sequence of the gRNA, i.e., that is sufficiently complementary to the guide sequence to permit specific binding of the guide sequence.
  • RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.
  • the specific length of the target sequence and the number of mismatches possible between the target sequence and the guide sequence depend, for example, on the identity of the Cas nuclease being directed by the gRNA.
  • a first sequence is considered to be “identical” or have “100% identity” with a second sequence if an alignment of the first sequence to the second sequence shows that all of the positions of the second sequence in its entirety are matched by the first sequence.
  • RNA and DNA generally the exchange of uridine for thymidine or vice versa
  • nucleoside analogs such as modified uridines
  • adenosine for all of thymidine, uridine, or modified uridine another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement.
  • sequence 5’-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU).
  • exemplary alignment algorithms are the Smith–Waterman and Needleman–Wunsch algorithms, which are well-known in the art.
  • a first sequence is considered to be “fully complementary” or 100% complementary” to a second sequence when all of the nucleotides of a first sequence are complementary to a second sequence, without gaps.
  • sequence UCU would be considered to be fully complementary to the sequence AAGA as each of the nucleobases from the first sequence base pair with the nucleotides of the second sequence, without gaps.
  • sequence UGU would be considered to be 67% complementary to the sequence AAGA as two of the three nucleobases of the first sequence base pair with nucleobases of the second sequence.
  • algorithms are available with various parameter settings to determine percent complementarity for any pair of sequences using, e.g., the NCBI BLAST interface (blast.ncbi.nlm.nih.gov/Blast.cgi) or the Needleman-Wunsch algorithm.
  • mRNA is used herein to refer to a polynucleotide that comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs).
  • mRNA can comprise one or more chemically modified nucleosides such as 5-methyl-cytidine (5mC), 2-thio-uridine (2sU),N1-methylpseudouridine (m1 ⁇ U) and pseudo-uridine ( ⁇ U), or a modified cap structure as provided below.
  • 5mC 5-methyl-cytidine
  • 2sU 2-thio-uridine
  • m1 ⁇ U 2-thio-uridine
  • ⁇ U pseudo-uridine
  • Exemplary guide sequences useful in the guide RNA compositions and methods described herein are shown in Tables 1A and 2A and throughout the application.
  • this guide sequence may be used in a guide RNA to direct a RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9, to a target sequence.
  • a RNA-guided DNA binding agent e.g., a nuclease, such as a Cas nuclease, such as Cas9
  • Target sequences as provided in Tables 1A and 2A by their positions within SEQ ID NO: 1, include both the positive and negative strands corresponding to the indicated DNA sequence in SEQ ID NO: 1 (i.e., the sequence given and the sequence’s reverse complement).
  • the guide sequence where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence, except for the substitution of U for T in the guide sequence.
  • “indels” refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in a target nucleic acid.
  • DSBs double-stranded breaks
  • inhibitor expression refers to a decrease in expression (e.g., knockdown or knockout) of a particular gene product (e.g., protein, mRNA, or both).
  • Expression of a protein i.e., gene product
  • a protein can be measured by detecting total cellular amount of the protein from a tissue sample, e.g., biopsy, or cell population of interest by detecting expression of a protein as individual members of a population of cells, e.g., by cell sorting to define percent of cells expressing a protein, or expression of a protein in cells in aggregate, e.g., by ELISA or western blot, including ELISA or western blot performed on a body fluid, e.g, blood or serum or plasma derived therefrom, into which the expressed protein is secreted.
  • a body fluid e.g, blood or serum or plasma derived therefrom
  • Inhibition of expression can result from a genetic modification in a regulatory sequence that is required for the expression of the gene product (e.g., a promoter sequence, a 3’ UTR sequence, a capping sequence, a 5’ UTR sequence, a poly A sequence, and the like). Inhibition of expression may also result from disrupting expression or activity of regulatory factors required for translation of the gene product, e.g., production of no gene product.
  • a genetic modification in a transcription factor sequence, inhibiting expression of the full-length transcription factor can have downstream effects and inhibit expression of one or more gene products controlled by the transcription factor. Therefore, inhibition of expression can be predicted by changes in genomic or mRNA sequences.
  • inhibit expression can be understood to inhibit expression of protein activity, even if the protein is expressed.
  • a mutation in a polymerase can inhibit activity of the polymerase without reducing the level of expression of the polymerase, e.g., as determined by a protein detection assay, e.g., ELISA or western blot.
  • inhibition of expression of an HBV polymerase can be determined by reduction of HBV polymerase activity, e.g., using methods provided herein, and does not necessarily require a lower level of protein as determined by a protein detection assay.
  • inhibition of protein activity does not require complete inhibition of protein activity.
  • inhibition of HBV polymerase activity can result in a decreased in HBV DNA, e.g., a therapeutically relevant decrease in HBV DNA.
  • Mutations expected to result in inhibition of expression can be detected by known methods including next generation sequencing of DNA isolated from a tissue sample or cell population of interest. Inhibition of expression can be determined as the percent of cells in a population having a predetermined level of expression of a protein, i.e., a reduction of the percent or number of cells in a population expressing a protein of interest at least a certain level. Inhibition of expression can also be assessed by determining a decrease in overall protein level, e.g., in a cell or tissue sample, e.g., a biopsy sample.
  • Mutations that disrupt protein function without a corresponding decrease in protein level may or may not be predictable from known sequences or structures and may need to be identified empirically.
  • inhibition of expression of a secreted protein can be assessed in a fluid sample, e.g., cell culture media or a body fluid. Proteins may be present in a body fluid, e.g., blood or urine, to permit analysis of protein level. In certain embodiments, protein level may be determined by protein activity or the level of a metabolic product, e.g., in urine or blood.
  • “inhibition of expression” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of an mRNA or a protein expressed in a tissue sample or by a population of cells. In some embodiments, “inhibition” may refer to some loss of expression of a particular gene product, for example at the cell surface or secreted into a bodily fluid, e.g., blood. In some embodiments, “inhibition” may refer to some loss of expression in one, or more, cell or tissue types, but not all cell or tissue types, e.g., inhibition of expression in liver, but not in other organs. It is understood that the level of inhibition of expression is relative to a starting level in the same type of subject sample.
  • routine monitoring of a protein level is more easily performed in a fluid sample from a subject, e.g., blood or urine, than in a tissue sample, e.g., a biopsy sample.
  • a correlation is known, or established, wherein the level of a biomarker, e.g., in blood or urine, is correlated with the level of inhibition of expression of a target gene. It is understood that the level of inhibition of expression is for the sample being assayed.
  • the target may be expressed in other tissues. Therefore, the level of inhibition of expression is not necessarily the level of inhibition of expression systemically, but within the tissue, cell type, or fluid being sampled.
  • a “genetic modification” is a change at the DNA level, e.g. induced by a CRISPR/Cas gRNA and Cas system, e.g., a CRISPR/Cas9 gRNA and Cas9 system.
  • a genetic modification may comprise an insertion, deletion, or substitution (i.e., base sequence substitution, i.e., mutation), typically within a defined sequence or genomic locus.
  • a genetic modification changes the nucleic acid sequence of the DNA.
  • a genetic modification may be at a single nucleotide position.
  • a genetic modification may be at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides.
  • the genetic modification may be at multiple overlapping ORF in the genome.
  • a genetic modification can be in a coding sequence, e.g., an open reading frame (ORF).
  • ORF open reading frame
  • a genetic modification can be used to prevent translation of an endogenous full-length protein having an amino acid sequence of the full-length protein prior to genetic modification of the defined sequence or genomic locus. Prevention of translation of a full-length protein or gene product includes prevention of translation of a protein or gene product of any length.
  • a genetic modification can include a modification that inhibits protein activity that may not correspondingly decrease protein level as determined by a protein detection assay, e.g., ELISA or western blot.
  • “Treatment” as used herein is understood as reducing at least one sign or symptom of the disease or indication. Reduction can include to a frequency or severity such that the sign or symptom of the disease is no longer detectable.
  • Treatment can include administration of more than one dose of the agent. For example, in some embodiments, treatment can include administration of 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.
  • Treatment can include administration with other agents. Effective treatment does not require a cure or complete elimination of the disease or indication.
  • the rate of progression or development of a disease can be compared to the progression or development of a disease in an appropriately matched control, e.g., a population control, a control from a natural history study.
  • the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation of one or more signs or symptoms in a subject with HBV infection including, but not limited to, the presence of serum HBV DNA or liver HBV ccc DNA, the presence of serum or liver HBV antigen, e.g., HBsAg or HBeAg.
  • Presence or levels of signs or symptoms can also be used to monitor therapeutic effects of treatment or determine a course of treatment (e.g., to determine the administration or amount of one or more doses or further doses).
  • a clinical marker e.g., serum HBsAg level
  • a subsequent dose is administered if the serum HBsAg is greater than a threshold value, optionally wherein the threshold value is 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment, or lower risk of HCC development.
  • treatment of HBV infection results in a “functional cure” of hepatitis B.
  • the term “functional cure” is understood to be clearance of circulating HBsAg and is preferably accompanied by conversion to a status in which HBsAg antibodies become undetectable using a clinically relevant assay.
  • undetectable antibodies can include a signal lower than 10 mIU/ml as measured by Chemiluminescent Microparticle Immunoassay (CMIA) or any other immunoassay, and may be called anti-HBs seroconversion.
  • Functional cure does not require clearance of all replicative forms of HBV (e.g., cccDNA from the liver).
  • Anti-HBs seroconversion occurs spontaneously in about 0.2-1% of chronically infected patients per year.
  • low level persistence of HBV is observed for decades indicating that a functional rather than a complete cure occurs. Without being bound to mechanism, it is proposed that the immune system is able to keep HBV in check.
  • a functional cure permits discontinuation of any treatment for the HBV infection.
  • a “functional cure” for HBV infection may not be sufficient to prevent or treat diseases or conditions that result from HBV infection, e.g., liver fibrosis, HCC, cirrhosis.
  • treatment of HBV infection results in a “sterilizing cure” of hepatitis B.
  • the term “sterilizing cure” is understood as clearance of all circulating HBsAg, is preferably accompanied by conversion to a status in which HBsAg antibodies become undetectable using a clinically relevant assay, and clearance of all detectable replicative forms of HBV (e.g., cccDNA from the liver) as determined by a clinically relevant assay (e.g., analysis of a liver biopsy).
  • delivering and “administering” are used interchangeably.
  • “Co-administration”, as used herein, means that a plurality of substances are administered sufficiently close together in time so that the agents act together.
  • Co- administration encompasses administering substances together in a single formulation and administering substances in separate formulations close enough in time so that the agents act together.
  • pharmaceutically acceptable means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable and that are not otherwise unacceptable for pharmaceutical use.
  • Pharmaceutically acceptable generally refers to substances that are non-pyrogenic.
  • Pharmaceutically acceptable can refer to substances that are sterile, especially for pharmaceutical substances that are for injection or infusion.
  • FIG. 1 is a graph showing percent editing of a hepatitis B coding sequence present in the HepAD38 cell line as determined by NGS.
  • Figure 3A and 3B are graphs showing percent C-to-T editing of a hepatitis B coding sequence present in theHepAD38 cell line as determined by NGS. Cells were contacted with guides targeting HBV S-Antigen at 2-fold serial dilutions from 100 nM to 1.56 nM, with a fixed concentration of mRNAs encoding (A) Spy-BC22n and UGI, or (B) Nme2-BC22n and UGI.
  • Figure 4A shows percent C-to-T editing against doses of Spy- BC22n guide RNAs.
  • Figure 4B shows percent C-to-T editing against doses of Nme2-BC22n guide RNAs.
  • Figure 5A and 5B show the percent knockdown of HBsAg levels relative to control guide RNA for Nme2 base editor guide RNAs.
  • Figure 5A shows the total intracellular HBsAg and the total amount of secreted HBsAg.
  • Figure 5B shows the percent intracellular HBV Copy Number (CN) knockdown for primer set 1 and primer set 2. Dotted line indicates the level of 90% knockdown.
  • Figure 6A and 6B show the percent knockdown of HBsAg levels relative to control guide RNA for Spy base editor guide RNA.
  • Figure 6A shows and total intracellular HBsAg and the total amount of secreted HBsAg.
  • Figure 6B shows the percent intracellular HBV Copy Number (CN) knockdown for primer set 1 and primer set 2.
  • Figure 7 shows the relative percent HBsAg knockdown relative to LNP-GFP mRNA in PHH. Guides were screened at 11, 33, or 100 ng/well.
  • Figure 8 shows the time course for the Log10 change of HBsAg level from the baseline over a period of 5 weeks post LNP injection using either Spy-BC22n or Nme2- BC22n guides.
  • Figure 9 shows the Log10 change in serum knockdown of total HBV DNA at five weeks post LNP injection with either Spy-BC22n or Nme2-BC22n guides.
  • Figure 10 shows the correlation between HBsAg and total HBV DNA levels in serum at five weeks post LNP injection relative to AAV only. Correlation is shown to be very high with an R 2 value of 0.9652.
  • Figure 11 shows the C-to-T editing as determined by NGS. Cells were treated with either Nme2-BC22n or Spy-BC22n guides targeting HBV in a dose response curve using a 3-fold dilution scheme with concentrations ranging from 50 nM to 0 nM.
  • Figure 12 shows the percent of secreted HBsAg knockdown using the two highest doses from the dose response curve of either a 50 nM or 16 nM dose of either Spy- BC22n or Nme2-BC22n guides.
  • Figure 13 shows the percent knockdown of secreted HBsAg in HepAD38 cells targeted by multiplex combinations of Nme2-BC22n HBV guides measured by MSD assay.
  • Figure 14 shows the percent knockdown of secreted HBsAg in HepAD38 cells targeted by additional multiplex combinations of Nme2-BC22n HBV guides measured by MSD assay.
  • Figure 15 shows the percent knockdown of secreted HBsAg with single or multiplexed HBV guides.
  • Figure 16 shows the percent total HBV DNA copy number knockdown with single or multiplexed HBV guides.
  • Figure 17 shows the HBsAg concentration (ng/ml) following treatment with guides with different modification patterns.
  • Figure 18 shows the mean HBsAg knockdown IC50 (nM) with guides with different modification patterns.
  • Figure 19 shows the serum HBsAg log10 reduction time course relative to individual animal baseline.
  • Figure 20 shows the log10 knockdown of total serum HBV DNA relative to TSS group at week 6.
  • Figure 21A shows the serum HBsAg log10 reduction time course relative to individual animal baseline with HBV genotype A.
  • Figure 21B shows the serum HBsAg log10 reduction time course relative to individual animal baseline with HBV genotype C.
  • Figure 22 shows the endpoint comparisons of log10 serum HBsAg reduction from baseline.
  • compositions comprising Guide RNA (gRNAs) [0221]
  • compositions useful for altering a DNA sequence e.g., inducing a single-stranded (SSB) or double-stranded break (DSB), within an HBV ORF, e.g., within nucleotides 163-466 of SEQ ID NO: 1, or also within nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, e.g., using a guide RNA with an RNA-guided DNA binding agent (e.g., a CRISPR/Cas system).
  • SSB single-stranded
  • DSB double-stranded break
  • a nucleic acid sequence of a representative HBV genome is provided as SEQ ID NO: 1: SEQ ID NO: 1 CAGTGGAATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTT CCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTAT CGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTC CTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACC GCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTG GCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCT GGTT
  • the #mer refers to the length of the crRNA or the sgRNA when a 20 nucleotide guide targeting sequence is included 5’ to the scaffold sequence provided in Table 3A.
  • Table 3A Exemplary Unmodified Spy Scaffold Sequences
  • Table 3B Exemplary Unmodified Spy Guide RNA Sequences Wherein the Ns collectively are a guide sequence provided herein.
  • the guide sequences may be integrated into the following modified guide scaffold motifs (Table 4).
  • the #mer refers to the length of the sgRNA when a 20 nucleotide guide targeting sequence, either a modified or unmodified sequence, is included 5’ to the scaffold sequence provided in Table 4:
  • Table 4 Exemplary Modified Spy Guide Scaffold Sequences ⁇ wherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and phosphodiesterase linkage.
  • the guide sequence is a chemically modified sequence.
  • the chemically modified guide sequence is (mN*)3(N)13- 17.
  • the guide sequence is (mN*)3(N)17, i.e., mN*mN*mN*NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN.
  • each N of the (N)13- 17 or the (N)17 is unmodified.
  • the each N in the (N)13-17 or the (N)17 is independently modified, e.g., independently modified with a 2’-O-methyl modification.
  • the guide sequences may further comprise a SpyCas9 sgRNA scaffold sequence.
  • SpyCas9 sgRNA scaffold sequence is shown in the Table 11 below (SEQ ID NO: 268: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGC – “Exemplary SpyCas9 sgRNA-1”), included at the 3’ end of the guide sequence, and provided with the domains as shown in the table below.
  • LS lower stem.
  • B is bulge.
  • US upper stem.
  • H1 and H2 are hairpin 1 and hairpin 2, respectively. Collectively H1 and H2 are referred to as the hairpin region.
  • gRNA is an sgRNA or a dgRNA, for example, and it optionally comprises a chemical modification.
  • the modified sgRNA comprises a guide sequence and a SpyCas9 sgRNA sequence, e.g., Exemplary SpyCas9 sgRNA-1.
  • a gRNA such as an sgRNA, may include modifications on the 5’ end of the guide sequence or on the 3’ end of the SpyCas9 sgRNA sequence, such as, e.g., Exemplary SpyCas9 sgRNA-1 at one or more of the terminal nucleotides, e.g., at 1, 2, 3, or 4 of the nucleotides at the 3’ end or at the 5’ end.
  • the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2- methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or an inverted abasic modified nucleotide; or a combination thereof.
  • the modified nucleotide includes a 2’-OMe modified nucleotide.
  • the modified nucleotide includes a PS linkage.
  • the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage.
  • SEQ ID NO: 268 GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGC “Exemplary SpyCas9 sgRNA-1,” see WO2019237069, the contents of which are incorporated herein by reference).
  • the portions of the Exemplary SpyCas9 sgRNA-1 and position numbering scheme are set forth in Table 11 below.
  • the Exemplary SpyCas9 sgRNA-1 further includes one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b.
  • nucleotides H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2.
  • the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 268); or 3.
  • the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or C.
  • Exemplary SpyCas9 sgRNA-1 SEQ ID NO: 268) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D.
  • the modified nucleotide is optionally selected from a 2’-O-methyl (2’- OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or a combination thereof; or 2.
  • the modified nucleotide optionally includes a 2’-OMe modified nucleotide.
  • Guide sequences targeted to sites adjacent to an appropriate PAM may further comprise additional nucleotides to form a crRNA or a crRNA joined to a trRNA to form a sgRNA e.g., with the exemplary nucleotide sequence following the guide sequence at its 3’ end as provided in Table 5.
  • the portions of the Exemplary NmeCas9 sgRNA and position numbering scheme, including both a guide sequence and a scaffold sequence, are set forth in Table 13 below.
  • the Exemplary NmeCas9 sgRNA-1 includes: A.
  • a guide RNA (gRNA) comprising a guide region and a conserved region, the conserved region comprising one or more of: (a) a shortened repeat/anti-repeat region, wherein the shortened repeat/anti- repeat region lacks 2-24 nucleotides, wherein (i) one or more of nucleotides 37-48 and 53-64 is deleted and optionally one or more of nucleotides 37-64 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2-10, optionally 2-8 nucleotides, wherein (i) one or more of nucle
  • exemplary unmodified conserved nucleotide sequences are shown in Table 5.
  • the #mer refers to the length of the sgRNA when a 24 nucleotide guide targeting sequence is included 5’ to the scaffold sequence provided in Table 5.
  • Table 5 Unmodified Nme conserveed Region Nucleotide Sequences
  • modified guide sequences may be integrated into one of the following exemplary modified conserved portion motifs (Table 6).
  • the #mer refers to the length of the sgRNA when a 24 nucleotide guide targeting sequence, either a modified or unmodified sequence, is included 5’ to the scaffold sequence provided in Table 6:
  • Table 6 Exemplary Modified conserveed Region Nme Guide RNA Motifs [0235] wherein “m” indicates a 2’-O-Me modification, and a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and a phosphorothioate linkage.
  • a targeting sequence is present on the 5’ end of the conserved portion of the guide RNA.
  • the targeting sequence is 20-25, preferably 22-24 nucleotides in length.
  • the guide targeting sequence comprises on or more chemical modifications, for example modifications at one or more of nucleotides 1, 2, and 3, optionally all of nucleotides 1, 2, and 3 at the 5’ end of the guide RNA.
  • the modification comprises a 2’-O-Me modification.
  • the modification comprises a 2’-O-Me modification and a phosphorothioate linkage to the 3’ nucleotide, e.g., (mN*)3(N)17-22, preferably (mN*)3(N)21, wherein each of the nucleotides in the (N)21 portion is independently modified or unmodified.
  • Exemplary SpyCas9 sgRNA-1, Exemplary NmeCas9 sgRNA-1, or an sgRNA, such as an sgRNA comprising an Exemplary SpyCas9 sgRNA-1 further includes a 3’ tail, e.g., a 3’ tail of 1, 2, 3, 4, or more nucleotides.
  • the tail includes one or more modified nucleotides.
  • the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a 2’ deoxy (2’H-) modified nucleotide, an abasic nucleotide, a locked nucleic acid (LNA) nucleotide, an unlocked nucleic acid (UNA) nucleotide, or a phosphorothioate (PS) linkage between nucleotides, a terminal inverted abasic modified nucleotide; or a combination thereof.
  • LNA locked nucleic acid
  • UNA unlocked nucleic acid
  • PS phosphorothioate
  • the modified nucleotide includes a 2’-OMe modified nucleotide. In certain embodiments, the modified nucleotide includes a PS linkage between nucleotides. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage between nucleotides. [0236] In certain embodiments, the hairpin region includes one or more modified nucleotides.
  • the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide; or a combination thereof.
  • the modified nucleotide includes a 2’-OMe modified nucleotide.
  • the upper stem region includes one or more modified nucleotides.
  • the modified nucleotide selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide; or a combination thereof.
  • the modified nucleotide includes a 2’-OMe modified nucleotide.
  • the Exemplary SpyCas9 sgRNA-1 or the Exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a modified nucleotide.
  • the modified nucleotide selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2- methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or a combination thereof.
  • the modified nucleotide includes a 2’-OMe modified nucleotide.
  • the Exemplary SpyCas9 sgRNA-1 or the Exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a sequence substituted nucleotide, wherein the pyrimidine is substituted for a purine.
  • the Watson-Crick based nucleotide of the sequence substituted pyrimidine nucleotide is substituted to maintain Watson-Crick base pairing.
  • the gRNA comprises one or more internal linkers.
  • internal linker describes a non-nucleotide segment joining two nucleotides within a guide RNA. If the gRNA contains a spacer region, the internal linker is located outside of the spacer region (e.g., in the scaffold or conserved region of the gRNA). For Type V guides, it is understood that the last hairpin is the only hairpin in the structure, i.e., the repeat-anti-repeat region.
  • the length of an internal linker may be dependent on, for example, the number of nucleotides replaced by the linker and the position of the linker in the gRNA.
  • the “bridging length” of an internal linker as used herein refers to the distance or number of atoms in the shortest chain of atoms on the pathway from the first atom of the linker (bound to a 3’ substituent, such as an oxygen or phosphate, of the preceding nucleotide to the last atom of the linker (bound to a 5’ substituent, such as an oxygen or phosphate) of the following nucleotide) (e.g., from ⁇ to # in the structure of Formula (I) described below). Approximate predicted bridging lengths for various linkers are provided in a table below.
  • L1 comprises one or more -CH 2 CH 2 O-, -CH 2 OCH 2 -, or -OCH 2 CH 2 - units (“ethylene glycol subunits”).
  • the number of -CH 2 CH 2 O-, -CH 2 OCH 2 -, or -OCH 2 CH 2 - units is in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • m is 1, 2, 3, 4 or 5.
  • m is 1, 2, or 3.
  • m is 6, 7, 8, 9, or 10.
  • L0 is null.
  • L0 is -CH 2 - or - CH 2 CH 2 -.
  • the internal linker substitutes for the loop in the nexus region of a Spy Cas9 gRNA corresponding to nucleotides 33-36 of SEQ ID NO: 268.
  • the internal linker is in a hairpin region of the gRNA.
  • the internal linker substitutes for at least 4 nucleotides of the hairpin region of the gRNA.
  • the internal linker substitutes for the loop in the hairpin 1 region of a Spy Cas9 gRNA, corresponding to nucleotides 53-56 in SEQ ID NO: 268.
  • the internal linker substitutes for the loop in the hairpin 1 region of an Nme Cas9 gRNA, corresponding to nucleotides 87-90 in SEQ ID NO: 455 and for at least 4 nucleotides the loop in the hairpin 2 region of an Nme Cas9 gRNA, corresponding to nucleotides 122-125 in SEQ ID NO: 455.
  • Table 9 Exemplary SpyCas9 guide RNAs comprising linkers
  • Nucleotide modifications in modified sequences are indicated in Table 9 as follows: wherein “m” indicates a 2’-O-Me modification, a “*” indicates a phosphorothioate linkage between nucleotides, and within the individually indicated nucleotides, no modification indicates an RNA (2’-OH) with a phosphodiesterase backbone.
  • (dS) denotes an abasic site having 1’,2’- dideoxyribose modification
  • (L3) denotes that the linker is S6, and (L4) denotes that the linker is S3.
  • Table 10 Exemplary NmeCas9 guide RNAs comprising linkers
  • Nucleotide modifications in modified sequences are indicated in Table 10 as follows: wherein “m” indicates a 2’-O-Me modification, a “*” indicates a phosphorothioate linkage between nucleotides, and within the individually indicated nucleotides, no modification indicates an RNA (2’-OH) with a phosphodiesterase backbone. Even in the context of a modified sequence, each nucleotide of (N)20-25 is optionally independently modified. In certain examples, at least the first three nucleotides are modified, e.g., (mN*)3(N)17-22.
  • a composition comprising one or more guide RNAs (gRNAs) comprising targeting sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in the HBV genome are provided.
  • gRNAs guide RNAs
  • a liver cell comprising a genetic modification in an HBV sequence, e.g., an HBV sequence integrated into the endogenous genome of the cell, or as cccDNA in the cytoplasm, within the target sites of HBV is provided.
  • an engineered cell comprising a genetic modification in an HBV sequence comprising a genetic modification in an HBV sequence
  • the genetic modification comprises a modification of at least one nucleotide within the genomic coordinates corresponding to an HBV targeting sequence selected from Tables 1A, 2A, 8, or 9, or that is complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides of nucleotides 163-466, 1-843, 1-1205, 2314- 3188, 1381-1846, or 1821-2460 of SEQ ID NO: 1.
  • an engineered cell comprising a genetic modification in an HBV sequence comprising a genetic modification in an HBV sequence
  • the genetic modification comprises a modification of at least one nucleotide within a target site, i.e., within a nucleotide sequence at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 20 contiguous nucleotides or that is at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 24 contiguous nucleotides of the exemplary target sites provided in Tables 1A and 2A, respectively. It is understood that the sequence of the HBV genome varies substantially across genotypes, and sometimes within individuals.
  • the guide RNA compositions provided herein are designed to recognize (e.g., hybridize to) a target sequence in an HBV gene, e.g., ORF.
  • the HBV target sequence may be recognized and cleaved, at one or both strands, by a provided Cas nuclease that is guided to the sequence by a guide RNA.
  • an RNA-guided DNA binding agent such as a Cas nuclease
  • a guide RNA may be directed by a guide RNA to a target sequence of an HBV gene, where the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-guided DNA binding agent, such as a Cas nuclease, cleaves the target sequence on one or both strands.
  • the selection of the one or more guide RNAs is determined based on target sequences within an HBV gene.
  • one or more guide RNAs are targeted to nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381- 1846, or 1821-2460 of SEQ ID NO: 1.
  • one or more guide RNAs are targeted to nucleotides 163-466 of SEQ ID NO: 1.
  • the guide RNA is targeted to nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240- 259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629- 648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899
  • the guide RNA is targeted to nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447- 470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1 wherein the guide RNA is an Nme Cas9 guide RNA.
  • mutations e.g., frameshift mutations resulting from indels, i.e., insertions or deletions, occurring as a result of a nuclease-mediated DSB, or sequence changes without a change in length, e.g., as a result of base editing
  • mutations in certain regions of the gene may be less tolerable than mutations in other regions of the gene, thus the location of a mutation is an important factor in the amount or type of protein knockdown that may result.
  • a base editor e.g., a deaminase
  • the use of a base editor, e.g., a deaminase, to change the nucleotide sequence in certain regions of the gene may be less tolerable than mutations in other regions of the gene, resulting in disruption of a start codon, insertion of a stop codon, or a change in a coding sequence for an amino acid essential in protein structure or function, e.g., polymerase structure or function.
  • a gRNA complementary or having complementarity to a target sequence within HBV is used to direct the RNA-guided DNA binding agent to a particular location in the appropriate HBV gene.
  • the Spy guide sequence is at least 90% or 95%; or 100% identical to the reverse complement of a target sequence present in an HBV gene.
  • the target sequence is complementary to the guide sequence of the guide RNA.
  • the degree of complementarity or identity between a guide sequence of a Spy guide RNA and its corresponding target sequence is at least 80%, 85%, preferably at least 90%, or 95%; or 100%.
  • the target sequence and the guide sequence of the Spy gRNA may be 100% complementary or identical.
  • the target sequence and the guide sequence of the Spy gRNA may contain at least one mismatch.
  • the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20.
  • the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides.
  • the Spy guide sequence comprises: (A) a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245- 264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629
  • the Nme guide sequence is at least 90% or 95%; or 100% identical to the reverse complement of a target sequence present in an HBV gene.
  • the target sequence may be complementary to the targeting sequence of the guide RNA.
  • the degree of complementarity or identity between a targeting sequence of an Nme guide RNA and its corresponding target sequence is at least 80%, 85%, preferably at least 90%, or 95%; or 100%.
  • the target sequence and the targeting sequence of the gRNA are100% complementary or identical.
  • the target sequence and the targeting sequence of the Nme gRNA may contain at least one mismatch to the target sequence in the HBV genome.
  • the target sequence and the targeting sequence of the gRNA may contain 1 or 2, less preferably 3, or 4 mismatches, where the total length of the guide sequence is 24. In some embodiments, the target sequence and the targeting sequence of the gRNA may contain 1-2 mismatches where the length of the guide sequence is 24 nucleotides.
  • the Nme targeting sequence comprises: (A) a sequence at least 80%, 85%, preferably at least 90%, or 95% identical to or complementary to 24 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951- 2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 7
  • a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein.
  • an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease is provided, used, or administered.
  • RNA-guided DNA binding agent; NmeCas9 [0265] RNA-guided DNA binding agents described herein encompass Neisseria meningitidis Cas9 (NmeCas9) and modified and variants thereof. In some embodiments, the NmeCas9 is Nme2 Cas9.
  • the NmeCas9 is Nme1 Cas9. In some embodiments, the NmeCas9 is Nme3 Cas9. [0266] Modified versions having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases.
  • compositions and methods comprise a nickase RNA-guided DNA binding agent, such as a nickase Cas, e.g., a nickase Cas9, that induces a nick rather than a double strand break in the target DNA.
  • a nickase Cas e.g., a nickase Cas9
  • the NmeCas9 nuclease may be modified to contain only one functional nuclease domain.
  • the RNA-guided DNA binding agent may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
  • a NmeCas9 nickase having a RuvC domain with reduced activity is used. In some embodiments, a NmeCas9 nickase having an inactive RuvC domain is used. In some embodiments, a NmeCas9 nickase having an HNH domain with reduced activity is used. In some embodiments, a NmeCas9 nickase having an inactive HNH domain is used. [0269] In some embodiments, a conserved amino acid within a NmeCas9 nuclease domain is substituted to reduce or alter nuclease activity. Wild type Cas9 has two nuclease domains: RuvC and HNH.
  • the RuvC domain cleaves the non-target DNA strand
  • the HNH domain cleaves the target strand of DNA.
  • the Cas9 nuclease comprises more than one RuvC domain or more than one HNH domain.
  • the Cas9 nuclease is a wild type Cas9.
  • the Cas9 is capable of inducing a double strand break in target DNA.
  • the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity.
  • a NmeCas9 may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include H588A (based on the N. meningitidis Cas9 protein).
  • the Cas protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include D16A (based on the NmeCas9 protein).
  • chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein.
  • a NmeCas9 nuclease domain may be replaced with a domain from a different nuclease such as Fok1.
  • a NmeCas9 protein is a modified NmeCas9 nuclease.
  • the nuclease is modified to induce a point mutation or base change, e.g., through deamination.
  • the Cas protein comprises a fusion protein comprising a Cas nuclease (e.g., NmeCas9), which is a nickase or is catalytically inactive, linked to a heterologous functional domain.
  • the Cas protein comprises a fusion protein comprising a catalytically inactive Cas nuclease (e.g., NmeCas9) linked to a heterologous functional domain (see, e.g., WO2014152432).
  • the catalytically inactive Cas9 is from the N. meningitidis Cas9.
  • the catalytically inactive Cas comprises mutations that inactivate the Cas.
  • the heterologous functional domain is a domain that modifies gene expression, histones, or DNA.
  • the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain.
  • the nuclease is a catalytically inactive Cas nuclease, such as dCas9.
  • the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase.
  • the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.
  • a heterologous functional domain such as a deaminase may be part of a fusion protein with a Cas nuclease having nickase activity or a Cas nuclease that is catalytically inactive discussed further below.
  • the Nme Cas9 has double stranded endonuclease activity.
  • the Nme Cas9 has nickase activity.
  • the Nme Cas9 comprises a dCas9 DNA binding domain.
  • the Nme Cas9 comprises an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 711, 716, 719, 722, or 730 (as shown in Table 40). In some embodiments, the Nme Cas9 comprises an amino acid sequence of any one of SEQ ID NOs: 711, 716, 719, 722, or 730 (as shown in Table 40).
  • the sequence encoding the NmeCas9 comprises a nucleotide sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 703, 704, 710, 715, 717-718, 720-721, or 728-729 (as shown in Table 40).
  • the sequence encoding the NmeCas9 comprises a nucleotide sequence of any one of SEQ ID NOs: 703, 704, 710, 715, 717-718, 720-721, or 728-729 (as shown in Table 40).
  • any of the foregoing levels of identity is at least 95%, 98%, or 99%; or 100%.
  • Exemplary polynucleotides and compositions comprising a deaminase and an RNA-guided nickase are provided as SEQ ID NOs: 701, 704, 706, 707, 715-722, or 728-730 in Table 40 below.
  • the RNA-guided DNA binding agent disclosed herein may further comprise a base-editing domain, such as a deaminase domain, that introduces a specific modification into a target nucleic acid.
  • a nucleic acid comprises an open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A), a C- terminal NmeCas9 nickase, and a first nuclear localization signal (NLS), wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI).
  • a second NLS is N-terminal to the NmeCas9 nickase.
  • the deaminase is N-terminal to an NLS (i.e., the first NLS or the second NLS).
  • the deaminase is N-terminal to all NLS in the polypeptide. In some embodiments, the deaminase is N-terminal to all NLS in the polypeptide, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI).
  • the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, a polypeptide encoded by the mRNA is provided.
  • the polypeptide comprises, from N to C terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase.
  • the polypeptide comprises, from N to C terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase.
  • NLS NLS
  • a cytidine deaminase e.g., APOBEC3A
  • an optional linker e.g., APOBEC3A
  • an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain e.g., a D16A Nme2Cas9 nickase.
  • the polypeptide comprises, from N to C terminus, first and second NLSs, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase.
  • a cytidine deaminase e.g., APOBEC3A
  • an optional linker e.g., APOBEC3A
  • an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain e.g., a D16A NmeCas9 nickase.
  • the polypeptide comprises, from N to C terminus, first and second NLSs, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase.
  • a cytidine deaminase e.g., APOBEC3A
  • an optional linker e.g., APOBEC3A
  • an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain e.g., a D16A Nme2Cas9 nickase.
  • the polypeptide comprises, from N to C terminus, A first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase.
  • a first NLS e.g., APOBEC3A
  • a second NLS e.g., an optional linker
  • an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain e.g., a D16A NmeCas9 nickase.
  • the polypeptide comprises, from N to C terminus, A first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase.
  • the polypeptide comprising A3A and an RNA-guided nickase does not comprise a uracil glycosylase inhibitor (UGI).
  • a composition comprising a first polypeptide, or an mRNA encoding a first polypeptide, comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI); and a second polypeptide, or an mRNA encoding a second polypeptide, comprising a uracil glycosylase inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide.
  • a cytidine deaminase which is optionally an APOBEC3A de
  • methods of modifying a target gene comprising administering the compositions described herein.
  • the method comprises delivering to a cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), and a second nucleic acid comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second nucleic acid is
  • the methods comprise delivering to a cell a polypeptide comprising a deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and a second NLS; wherein the first NLS and the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the polypeptide, and delivering to the cell a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the UGI.
  • a deaminase which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NL
  • a molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is from about 1:35 to from about 30:1. In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is not about 1:1.
  • the composition described herein further comprises at least one gRNA. In some embodiments, the composition described herein further comprises two gRNAs. In some embodiments, a composition is provided that comprises an mRNA described herein and at least one gRNA, e.g., two gRNAs. In some embodiments, the gRNA is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • the gRNA is a dual guide RNA (dgRNA).
  • the composition is capable of effecting genome editing upon administration to the subject.
  • Cytidine deaminase; APOBEC3A Deaminase encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol.
  • the cytidine deaminase disclosed herein is an enzyme of APOBEC family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC3 subgroup.
  • A3A variants share homology to wild-type A3A, or a fragment thereof.
  • a A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to a wild type A3A.
  • the A3A variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type A3A.
  • the A3A variant comprises a fragment of an A3A, such that the fragment has at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of a wild-type A3A.
  • an A3A variant is a protein having a sequence that differs from a wild-type A3A protein by one or several mutations, such as substitutions, deletions, insertions, one or several single point substitutions.
  • a shortened A3A sequence could be used, e.g.
  • an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild- type sequence).
  • the wild-type A3A is a human A3A (UniPROT accession ID: p31941, SEQ ID NO: 734).
  • the A3A disclosed herein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 734.
  • the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
  • the A3A comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 734.
  • the A3A comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 734.
  • the A3A comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 99% identity to A3A ID NO: 734. In some embodiments, the A3A comprises the amino acid sequence of SEQ ID NO: 734. [0302] In some embodiments, the cytidine deaminase disclosed herein comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 734-216735.
  • any of the foregoing levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
  • the UGI comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 709 or 735.
  • the polypeptide comprising the deaminase and the RNA-guided nickase described herein further comprises a linker that connects the deaminase and the RNA-guided nickase.
  • the linker is a peptide linker.
  • the nucleic acid encoding the polypeptide comprising the deaminase and the RNA-guided nickase further comprises a sequence encoding the peptide linker.
  • mRNAs encoding the deaminase-linker-RNA-guided nickase fusion protein are provided.
  • the peptide linker is any stretch of amino acids having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids.
  • the peptide linker is the 16 residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)).
  • the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 652), SGSETPGTSESA (SEQ ID NO: 653), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 654).
  • the peptide linker comprises a (GGGGS)n (SEQ ID NO: 655), a (G)n (SEQ ID NO: 823), an (EAAAK)n(SEQ ID NO: 656), a (GGS)n (SEQ ID NO: 824), an SGSETPGTSESATPES (SEQ ID NO: 657) motif (see, e.g., Guilinger J P, Thompson D B, Liu D R.
  • the peptide linker comprises one or more sequences selected from SEQ ID NOs: 652-657.
  • the gRNA is chemically modified.
  • a gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.
  • a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.”
  • Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); and (iv) modification of the 3' end or 5' end of the oligonucleotide to provide exonuclease stability, e.g
  • modified gRNAs or mRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications.
  • a modified residue can have a modified sugar and a modified nucleobase.
  • up to 15% of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups.
  • modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA.
  • modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA.
  • the gRNA comprises one, two, three or more modified residues.
  • at least 5% (e.g., at least 5%, 10%, 15%, preferably at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the positions in a modified gRNA are modified nucleosides or nucleotides.
  • at least 5% of the positions in the modified guide RNA are modified nucleotides or nucleosides.
  • at least 10% of the positions in the modified guide RNA are modified nucleotides or nucleosides.
  • at least 15% of the positions in the modified gRNA are modified nucleotides or nucleosides.
  • At least 20% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments, no more than 65% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 55% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 50% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 10-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-70% of the positions in the modified gRNA are modified nucleotides.
  • nuclease is a Spy Cas9 nuclease. In some embodiments, range 30-70% of the positions in the modified gRNA are modified nucleotides and the nuclease is an Nme Cas9 nuclease.
  • Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds.
  • the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases.
  • the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo.
  • the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
  • the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent.
  • the modified residue e.g., modified residue present in a modified nucleic acid
  • the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
  • modified phosphate groups include, phosphorothioate, borano phosphate esters, methyl phosphonates, phosphoroamidates, phosphodithioate, alkyl or aryl phosphonates and phosphotriesters.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • the backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • the replacement can occur at either linking oxygen or at both of the linking oxygens.
  • the phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications, e.g., an amide linkage.
  • the charged phosphate group can be replaced by a neutral moiety.
  • moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, carboxymethyl, carbamate, amide, thioether.
  • moieties which can replace the phosphate group can include, without limitation, e.g., ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
  • Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications.
  • the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • PNA peptide nucleic acid
  • the modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification.
  • the 2' hydroxyl group can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents.
  • modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'- alkoxide ion.
  • Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH 2 CH 2 O)nCH 2 CH 2 OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20).
  • R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar
  • PEG polyethyleneg
  • the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride.
  • the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C 1-6 alkylene or C 1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH 2 ) n -amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheter
  • the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond.
  • the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH 2 CH 2 OCH 3 , e.g., a PEG derivative).2' modifications can include hydrogen (i.e.
  • deoxyribose sugars ); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH 2 CH 2 NH) n CH 2 CH 2 - amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g.
  • the sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar.
  • the modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms.
  • the modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides. As used herein, a single abasic sugar is not understood to result in a discontinuity of a duplex.
  • 2’ modifications include, for example, modifications include 2’-OMe, 2’-F, 2’-H, optionally 2’-O-Me.
  • the modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid can include a modified base, also called a nucleobase.
  • nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uridine (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids.
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog.
  • the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
  • each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA or tracr RNA.
  • one or more residues at one or both ends of the sgRNA may be chemically modified, or internal nucleosides may be modified, or the sgRNA may be chemically modified throughout. Certain embodiments comprise a 5' end modification.
  • the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018/107028, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2017/136794, the contents of which are hereby incorporated by reference in their entirety.
  • the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2019/237069, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2021/119275, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in Nme guides, the contents of which are hereby incorporated by reference in their entirety.
  • the sgRNA comprises any of the modification patterns shown herein, where N is any natural or non-natural nucleotide, and wherein the totality of the N’s comprise an HBV guide sequence as described herein in Table 1A.
  • the modified sgRNA comprises a sequence shown in Table 15 or in Table 9. Table 15: Modified Spy Guide RNA Sequences
  • N’s comprise a targeting sequence comprising (A) a sequence at least 80%, 85%, preferably 90% or 95% identical to or complementary to at least 20 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381- 1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269
  • the N’s are replaced with any of the guide sequences disclosed herein in Table 1A.
  • each N of the N17 may be independently modified, e.g., modified with a 2’-OMe modification.
  • the sgRNA comprises a sequence wherein the totality of the N’s comprise an HBV targeting sequence as described herein in Table 2A.
  • the modified sgRNA comprises a sequence provided in Table Table 16 or Table 10: Table 16: Exemplary Modified Nme Guide RNA Motifs
  • a targeting sequence is present on the 5’ end of the conserved portion of the guide RNA.
  • the targeting sequence is 20-25, preferably 22-24 nucleotides in length.
  • the guide targeting sequence comprises on or more chemical modifications, for example modifications at one or more of nucleotides 1, 2, and 3, optionally all of nucleotides 1, 2, and 3 at the 5’ end of the guide RNA.
  • the modification comprises a 2’-O- Me modification. In certain embodiments, the modification comprises a 2’-O-Me modification and a phosphorothioate linkage to the 3’ nucleotide, e.g., (mN*)3(N)17-22, preferably (mN*)3(N)21, wherein each of the nucleotides in the (N)21 portion is independently modified or unmodified.
  • the (N)20-25 has the following sequence and modification pattern mN*mN*mN*mN*mNmNNNmNmNNmNNNmNNmNNmNNNmNNNmNNNmNNN.
  • the totality of N’s comprise an HBV targeting sequence comprising: (A) a sequence at least 80%, 85%, preferably at least 90%, or 95% identical, or 100% identical to or complementary to 24 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460of SEQ ID NO: 1; (2) nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713- 736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-3
  • each N of the (N)20-25 may be independently modified, e.g., modified with a 2’-OMe modification, optionally further with a PS modification, particularly at 1, 2, or 3 terminal nucleotides.
  • the (N)20-25 has the following sequence and modification pattern mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN. [0326] Any of the modifications described below may be present in the gRNAs and mRNAs described herein.
  • RNA nucleotide i.e., 2’-OH with a phosphodiesterase linkage to the 3’ nucleotide.
  • mA RNA nucleotide
  • mU adenine, cytosine, uridine, or guanidine nucleotide, respectively, that has been modified with 2’-O-Me.
  • Modification with 2’-O-methyl can be depicted as follows: [0330] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution.
  • 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.
  • the terms “fA,” “fC,” “fU,” or “fG” are used to denote a nucleotide that has been substituted with 2’-F.
  • Substitution of 2’-F can be depicted as follows: [0333] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one non-bridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases.
  • PS Phosphorothioate
  • the modified oligonucleotides may also be referred to as S-oligos.
  • a “*” is used to denote a PS modification.
  • the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a PS bond.
  • mA* mC*
  • mU* mU*
  • mG* a nucleotide that has been substituted with 2’-O-Me and that is linked to the next (e.g., 3’) nucleotide with a PS bond.
  • Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage). Such inverted bases can only be present as a terminal nucleotide. In chemical synthesis methods performed 3’ to 5’, inverted bases do not have 5’ hydroxy available to grow the chain. For example:
  • An abasic nucleotide can be attached with an inverted linkage.
  • an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage.
  • An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap.
  • one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified.
  • the modification is a 2’-O-Me, 2’-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability or performance.
  • the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.
  • PS phosphorothioate
  • the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise an inverted abasic nucleotide. [0343] In some embodiments, the guide RNA comprises a modified sgRNA.
  • the guide sequence comprises a sequence of any one of SEQ ID NOs: 95-192 shown in Table 2A.
  • a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease, as described herein.
  • an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease is provided, used, or administered.
  • the ORF encoding an RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified.
  • the mRNA or modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions.
  • the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl.
  • the modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof.
  • the modified uridine is 5-methoxyuridine.
  • the modified uridine is 5-iodouridine.
  • the modified uridine is pseudouridine.
  • the modified uridine is N1-methyl- pseudouridine.
  • the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5- methoxyuridine.
  • an mRNA disclosed herein comprises a 5’ cap, such as a Cap0, Cap1, or Cap2.
  • a 5’ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5’- triphosphate to the 5’ position of the first nucleotide of the 5’-to-3’ chain of the mRNA, i.e., the first cap-proximal nucleotide.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-hydroxyl.
  • the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2’-methoxy and a 2’-hydroxyl, respectively.
  • the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115.
  • Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2.
  • Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon.
  • Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA.
  • a cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No.
  • AM8045 is a cap analog comprising a 7- methylguanine 3’-methoxy-5’-triphosphate linked to the 5’ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation.
  • ARCA results in a Cap0 cap in which the 2’ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘anti- reverse’ cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG,” RNA 7: 1486–1495.
  • CleanCap TM AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap TM GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally.3’-O-methylated versions of CleanCap TM AG and CleanCap TM GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively, or CleanCap AU: TriLink Biotechnologies as Cat. Nos. N-7114.
  • a cap can be added to an RNA post-transcriptionally.
  • Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit.
  • it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad.
  • the mRNA further comprises a poly-adenylated (poly- A) tail.
  • the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines.
  • the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides.
  • the poly-A tail includes non-adenine nucleotides, i.e., is an interrupted poly-A tail.
  • the poly-A tail is interrupted by a non-adenine nucleotide about every 40, 50, 60, 70, 80, or 90 nucleotides. In certain embodiments, the poly-A tail is interrupted by a non- adenine nucleotide about every 50 nucleotides.
  • Ribonucleoprotein complex [0357] In some embodiments, a composition is encompassed comprising one or more sgRNAs comprising one or more guide sequences from Table 1A or one or more sgRNAs from Table 2A and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9.
  • the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity.
  • the RNA-guided DNA-binding agent comprises a Cas nuclease.
  • Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, Neisseria meningitidis, and other prokaryotes as known in the art , and modified (e.g., engineered or mutant) versions thereof.
  • the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes.
  • the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis.
  • the gRNA together with an RNA-guided DNA binding agent is called a ribonucleoprotein complex (RNP).
  • the RNA-guided DNA binding agent is a Cas nuclease.
  • the gRNA together with a Cas nuclease is called a Cas RNP.
  • the RNP comprises Type-I, Type-II, or Type-III components.
  • the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system.
  • the gRNA together with Cas9 is called a Cas9 RNP.
  • Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA.
  • the Cas9 protein comprises more than one RuvC domain or more than one HNH domain.
  • the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA.
  • chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein.
  • a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1.
  • a Cas nuclease may be a modified nuclease.
  • the Cas nuclease may be from a Type-I CRISPR/Cas system.
  • the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system.
  • the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity. [0363] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase.
  • a nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix.
  • a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., US Pat. No.8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations.
  • a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.
  • the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain.
  • the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
  • a nickase is used having a RuvC domain with reduced activity.
  • a nickase is used having an inactive RuvC domain.
  • a nickase is used having an HNH domain with reduced activity.
  • a nickase is used having an inactive HNH domain.
  • a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity.
  • a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771.
  • the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015).
  • meningitidis include Nme2Cas9D16A (HNH nickase) and Nme2Cas9H588A (RuvC nickase) [0366]
  • an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively.
  • the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking).
  • use of double nicking may improve specificity and reduce off-target effects.
  • a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.
  • the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity.
  • the dCas polypeptide is a dCas9 polypeptide.
  • the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 20140186958; US 20150166980; and US 20190338308.
  • the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).
  • the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell.
  • the heterologous functional domain may be a nuclear localization signal (NLS).
  • the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, or 4 NLS(s).
  • the RNA-guided DNA-binding agent may be fused with two NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. In some embodiments, the NLS is not linked to the C-terminus. It may also be inserted within the RNA-guided DNA binding agent sequence. In certain circumstances, at least the two NLSs are the same (e.g., two SV40 NLSs). In certain embodiments, at least two different NLSs are present the RNA-guided DNA binding agent.
  • the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. [0370] SV40 NLS, PKKKRKV (SEQ ID NO: 679) or PKKKRRV (SEQ ID NO: 680).
  • the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 681).
  • the NLS sequence may comprise LAAKRSRTT (SEQ ID NO: 682), QAAKRSRTT (SEQ ID NO: 683), PAPAKRERTT (SEQ ID NO: 684), QAAKRPRTT (SEQ ID NO: 685), RAAKRPRTT (SEQ ID NO: 686), AAAKRSWSMAA (SEQ ID NO: 687), AAAKRVWSMAF (SEQ ID NO: 688), AAAKRSWSMAF (SEQ ID NO: 689), AAAKRKYFAA (SEQ ID NO: 690), RAAKRKAFAA (SEQ ID NO: 691), or RAAKRKYFAV (SEQ ID NO: 692).
  • the NLS may be a snurportin-1 importin- (IBB domain, e.g. an SPN1-imp sequence. See Huber et al., 2002, J. Cell Bio., 156, 467-479.
  • a single PKKKRKV (SEQ ID NO: 693).
  • the first and second NLS are independently selected from an SV40 NLS, a nucleoplasmin NLS, a bipartite NLS, a c-myc like NLS, and an NLS comprising the sequence KTRAD (SEQ ID NO: 820).
  • the first and second NLSs may be the same (e.g., two SV40 NLSs).
  • the first and second NLSs may be different.
  • the first NLS is a SV40NLS and the second NLS is a nucleoplasmin NLS.
  • the SV40 NLS comprises a sequence of PKKKRKVE (SEQ ID NO: 694) or KKKRKVE (SEQ ID NO: 695).
  • the nucleoplasmin NLS comprises a sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 696).
  • the bipartite NLS comprises a sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 697).
  • the c-myc like NLS comprises a sequence of PAAKKKKLD (SEQ ID NO: 698).
  • One or more linkers are optionally included at the fusion site of the NLS to the nuclease, or between NLS when more than one is present.
  • one or more NLS(s) according to any of the foregoing embodiments are present in the RNA-guided DNA-binding agent in combination with one or more additional heterologous functional domains.
  • One or more linkers are optionally included at the fusion site.
  • the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent.
  • the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases.
  • proteolytic enzymes such as, for example, proteasomes, lysosomal proteases, or calpain proteases.
  • the heterologous functional domain may comprise a PEST sequence.
  • the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain.
  • the ubiquitin may be a ubiquitin-like protein (UBL).
  • ULB ubiquitin-like protein
  • Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal- precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S.
  • the heterologous functional domain may be a marker domain.
  • marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences.
  • the marker domain may be a fluorescent protein.
  • Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1 ), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, H
  • the marker domain may be a purification tag or an epitope tag.
  • Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis (SEQ ID NO: 821), 8xHis (SEQ ID NO: 822), biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • MBP maltose binding protein
  • TRX thioredoxin
  • Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
  • the heterologous functional domain may be an effector domain.
  • the effector domain may modify or affect the target sequence.
  • the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No.9,023,649.
  • the heterologous functional domain is a transcriptional activator or repressor.
  • the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.
  • the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase.
  • the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.
  • the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP.
  • the gRNA is expressed together with an RNA-guided DNA binding agent, such as a Cas protein, e.g. Cas9.
  • the gRNA is delivered to or expressed in a cell line that already stably expresses an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • the gRNA is delivered to a cell as part of a RNP.
  • the gRNA is delivered to a cell along with a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • an RNA-guided DNA nuclease such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase.
  • RNA-guided DNA nuclease results in a base change that results in a premature stop codon, disruption of a start codon or regulatory sequence, or introduces a mutation that disrupts function of the encoded protein, or a combination thereof.
  • the efficacy of particular gRNAs is determined based on in vitro models.
  • the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9, e.g., available from ATCC).
  • the cell line is a HepAD38 cell line.
  • the in vitro model is a primary cell line, e.g., a primary liver cell line, e.g., primary hepatocytes.
  • the in vitro model is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the in vitro model is a T cell, such as primary human T cells. With respect to using primary cells, commercially available primary cells can be used to provide greater consistency between experiments.
  • the number of off-target sites at which a deletion or insertion occurs in an in vitro model is determined, e.g., by analyzing genomic DNA from transfected cells in vitro with Cas9 mRNA and the guide RNA.
  • such a determination comprises analyzing genomic DNA from the cells transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide. Exemplary procedures for such determinations are provided in the working examples in which various cell types are used.
  • the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process.
  • a cell line comparison of data with selected gRNAs is performed. In some embodiments, cross screening in multiple cell models is performed. [0381] In some embodiments, the efficacy of a guide RNA is measured by percent indels or percent genetic modifications of the HBV genome sequence, either integrated into the genome of the host cell or as cccDNA in the cytoplasm. In some embodiments, the efficacy of a guide RNA is measured by percent indels or percent genetic modifications at an HBV genome locus, e.g., HepAD38 (Genotype D) ( see, e.g., Ladner SK et al.1997, AAC 41:1715–1720).
  • HepAD38 Geneotype D
  • the efficacy of a guide RNA is measured by percent indels or percent genetic modifications of an HBV target site of Tables 1A and 2A. In some embodiments, the percent editing of an HBV genome locus compared to the percent indels or genetic modifications necessary to achieve reduction, e.g., knockdown, of the HBV protein products. In some embodiments, the efficacy of a guide RNA is measured by reduced expression of an HBV protein. In embodiments, said reduced expression of a protein is as measured by ELISA, e.g., as described herein. In certain embodiments, the protein is a secreted protein, e.g., in cell culture media or in blood or a fraction derived therefrom, e.g., serum.
  • the HBV protein expression is reduced using the methods and compositions disclosed herein.
  • the level of protein as determined, e.g., by ELISA is reduced by at least 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, or 99% relative to a control population of unmodified cells.
  • the level of protein is determined by a protein activity assay, e.g., a polymerase assay.
  • an “unmodified cell” refers to a control cell (or cells) of the same type of cell in an experiment or test, wherein the “unmodified” control cell has not been contacted with an HBV guide. Therefore, an unmodified cell (or cells) may be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that does not target HBV. As used herein, an “unmodified cell” can include the HepAD38 cell line that has not been contacted with an HBV guide.
  • the efficacy of a guide RNA is measured by the number or frequency of indels or genetic modifications at off-target sequences within the genome of the target cell type, such as a primary hepatocyte cell.
  • efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g., ⁇ 5%) in a cell population or relative to the frequency of indel creation at the target site.
  • the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., a primary hepatocyte cell), or which produce a frequency of off-target indel formation of ⁇ 5% in a cell population or relative to the frequency of indel creation at the target site.
  • the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., primary hepatocyte cell) as compared to a control cell.
  • guide RNAs are provided which produce indels at less than 5 validated off-target sites.
  • guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 validated off-target site(s).
  • the off-target site(s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome.
  • the efficacy of a guide RNA is measured in vivo, e.g., in an animal or animal model having a DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, i.e., having a DNA sequence sufficiently complementary to the targeting sequence in the guide RNA proximal to a cognate PAM for the guide and nuclease.
  • the animal has an endogenous DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA.
  • the animal model is a transgenic model, e.g., a mouse model having an inserted DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, e.g., a mouse having an inserted DNA sequence, e.g., a viral DNA sequence or a human DNA sequence, e.g. a sequence from a human albumin.
  • the inserted sequence may or may not include one or more intron sequence or regulatory sequence, e.g., 3’ UTR, 5’ UTR, present in the human gene in its native context.
  • the human DNA sequence may replace the homologous endogenous DNA sequence
  • the mouse albumin gene is replaced by the human albumin gene.
  • the human gene is present in the mouse in the context of a human hepatocyte, e.g., a mouse with a humanized liver, e.g., as available from PhoenixBio or Inotiv.
  • the inserted DNA sequence is a viral DNA sequence in a human hepatocyte in a mouse with a humanized liver.
  • the inserted DNA sequence is a viral sequence containing the target site inserted into a viral vector of a different virus, e.g., an HBV DNA sequence in an AAV vector.
  • detecting gene editing events such as the formation of insertion/deletion (“indel”) mutations and insertion or homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (herein after referred to as “LAM-PCR,” or “Linear Amplification (LA)” method).
  • LAM-PCR Linear Amplification
  • detecting gene editing events that are base editing events e.g., changes in nucleotide sequence without insertion or deletion of nucleotides, are detected by next generation sequencing (NGS).
  • NGS next generation sequencing
  • the efficacy of a guide RNA is measured by the levels of functional protein or protein complexes comprising the expressed protein product of the gene, e.g., polymerase activity assay. In some embodiments, the efficacy of a guide RNA is measured by ELISA. Genetic modification for inhibition of target gene expression [0387]
  • the cells or population of cells comprise a genetic modification, e.g., of a nucleic acid sequence encoding an HBV gene, e.g., integrated into the host genome or as cccDNA present in the cytoplasm.
  • the engineered cells or population of cells comprise a genetic modification of an HBV gene as assessed by sequencing, e.g., NGS, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90% 95%, or 99% of cells comprise an insertion, deletion, or substitution in the HBV sequence. It is understood that there are multiple HBV genotypes, and that genetic modifications are compared to the HBV sequence present in the cell which may or may not be different from the HBV sequence, SEQ ID NO: 1, provided herein. In some embodiments, at least 50% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • At least 55% of cells in the population comprise a modification.
  • at least 60% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • at least 65% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. on selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • at least 70% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • at least 75% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • At least 80% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 85% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some preferred embodiments, at least 90% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 95% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 99% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence.
  • HBV protein expression e.g., HBsAg protein expression
  • HBsAg protein expression is decreased by at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90%, 95%, or 99% as compared to a level wherein the HBV gene has not been modified.
  • expression of HBsAg is decreased by at least 70%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene, has not been modified.
  • expression of HBsAg is decreased by at least 85%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 90%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified.
  • expression of HB P is decreased by at least 75%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is decreased by at least 80%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified.
  • expression of HB P is decreased by at least 85%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is preferably decreased by at least 90%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified.
  • expression of HB P is decreased by at least 95%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene, has not been modified. In some embodiments, expression of HB P is decreased by at least 99%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is decreased by no more than 95%, as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified.
  • expression of HB P is decreased by no more than 99%, as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified.
  • Assays for HB P protein expression and activity, and mRNA expression are known in the art.
  • HBV protein expression e.g., HBsAg expression, e.g., as demonstrated by HBsAg level
  • HB P expression e.g., as demonstrated by protein level or activity
  • HBsAg expression and HB P expression are both reduced by use of one gRNA targeted to a single site in within overlapping region of the HBsAg and HB P ORFs, e.g., within the HBsAg ORF and the HB P ORF in SEQ ID NO: 1. It is understood that due to the variation in HBV genomes, a gRNA that is effective in targeting both the HBsAg ORF and the HB P ORF in SEQ ID NO: 1 may not be useful in targeting both ORFs in all HBV genotypes.
  • HBsAg expression and HB pol expression are reduced by use of at least two gRNA targeted to distinct sites, e.g., non-overlapping sites, in the HBsAg ORF and HB pol ORFs.
  • the level of expression may be inhibited in one, but not in all, tissues where the target gene is expressed.
  • the level of expression of an HBV protein may be inhibited in one, but not all, HBV genotypes in which the ORFs are expressed.
  • surrogate markers can be used to monitor changes in expression.
  • the level of inhibition of expression may be determined by or correlated to a decrease in levels in protein in the blood.
  • inhibition of expression in the liver can result in a change in a metabolite or other biomarker in a body fluid, e.g., blood or urine.
  • the change in the level of the metabolite can be correlated with the level of inhibition of expression.
  • Such correlations can be useful for monitoring the level of inhibition of expression as serial biopsies, e.g., serial liver biopsies, are not practical for monitoring in human subjects, or often in animal models.
  • the target gene is genetically modified resulting in inhibition of expression in a cell using a guide RNA with an RNA-guided DNA binding agent.
  • a break e.g., double-stranded break (DSB) or single-stranded break (nick)
  • a guide RNA with an RNA-guided DNA-binding agent e.g., a CRISPR/Cas system.
  • the methods may be used in vitro, e.g., for screening guides, or in vivo, e.g., to provide a therapeutic benefit.
  • the guide RNAs mediate a target-specific cutting of one or both DNA strands by an RNA-guided DNA-binding agent (e.g., Cas nuclease) at a site described herein within a target gene.
  • an RNA-guided DNA-binding agent e.g., Cas nuclease
  • the guide RNAs comprise guide sequences that bind to, or are capable of binding to, those regions.
  • IV. Methods and Uses Including Therapeutic Methods and Uses of Genome Editing Agents [0396] The gRNAs and associated methods and compositions disclosed herein are useful for making genome editing therapeutic agents.
  • the gRNAs comprising the guide sequences of Tables 1A and 2A together with an RNA-guided DNA nuclease such as a Cas nuclease induce breaks, either single stranded breaks or double stranded breaks. Double stranded breaks lead to non-homologous ending joining (NHEJ) during repair, leading to a modification, e.g., a mutation, in an HBV gene.
  • NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in an HBV gene.
  • Single stranded breaks can be coupled to base editing resulting in a sequence change, e.g., generating a stop codon, disrupting a start site, or a mutation that disrupts function of the ORF.
  • gRNAs comprising guide sequences targeted to target genomic sequences are also delivered to the cell together with RNA-guided DNA nuclease such as a Cas nuclease, either together or separately, to make a genetic modification in a target genomic sequence to inhibit the expression of a full-length expression product from the target gene. Due to the overlapping nature of the HBV genome, one guide RNA may cause changes in more than one ORF in the HBV genome.
  • the gRNAs are sgRNAs.
  • the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse in which the liver has been repopulated with human hepatocytes.
  • the guide RNAs, compositions, and formulations are used to produce a cell in vivo, e.g., liver cell, e.g., a hepatocyte with a genetic modification in an HBV gene, that may be integrated into the host genome or present as an episomal copy, e.g., as cccDNA.
  • Lipid nanoparticles are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs and compositions disclosed herein in vivo and in vitro.
  • the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.
  • a method for delivering any one of the cells or populations of cells disclosed herein to a subject is provided, wherein the gRNA is delivered via an LNP in vivo.
  • the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9.
  • a composition comprising any one of the gRNAs disclosed and an LNP is provided.
  • the composition further comprises a Cas9 or an mRNA encoding Cas9.
  • the composition further comprises a uracil glycosylase inhibitor (UGI) or an mRNA encoding a UGI.
  • UGI uracil glycosylase inhibitor
  • LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder.
  • a method for delivering any one of the gRNAs disclosed herein in vivo is provided, wherein the gRNA is associated with an LNP.
  • the gRNA is not associated with an LNP.
  • the gRNA/LNP or gRNA is also associated with a Cas9 or an mRNA encoding Cas9.
  • the gRNA/LNP, gRNA, Cas9, or mRNA encoding a Cas9 is also associated with a UGI or an mRNA encoding a UGI.
  • the guide RNA compositions described herein, alone or encoded on one or more vectors are formulated in or administered via a lipid nanoparticle; see e.g., WO2017/173054 and WO2021/222287, the contents of each of which are hereby incorporated by reference in their entirety.
  • DNA or RNA vectors encoding any of the guide RNAs comprising any one or more of the guide sequences described herein are provided.
  • the vectors further comprise nucleic acids that do not encode guide RNAs.
  • Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9.
  • the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA.
  • the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9.
  • the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9.
  • the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9).
  • the Cas9 is from Neisseria meningitidis (i.e., Nme Cas9).
  • the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system.
  • the nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.
  • the components can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or they can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus).
  • viral vectors e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus.
  • Methods and compositions for non-viral delivery of nucleic acids include lipofection, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acid (e.g., naked DNA/RNA), artificial virions, and agent-enhanced uptake of DNA.
  • Hepatitis B virus used interchangeably with the term “HBV” refers to the well-known non-cytopathic, liver-tropic DNA virus belonging to the Hepadnaviridae family.
  • HBV genome is partially double-stranded, circular DNA with overlapping reading frames.
  • genes that may be referred to herein as “genes” or “open reading frames” based on size, encoded by the HBV genome. These contain open reading frames called C, X, P, and S.
  • the core protein is coded for by gene C (HBcAg).
  • Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein.
  • the DNA polymerase is encoded by gene P.
  • Gene S is the gene that codes for the surface antigens (HBsAg).
  • the HBsAg gene is one long open reading frame which contains three in frame “start” (ATG) codons resulting in polypeptides of three different sizes called large, middle, and small S antigens, pre-S1+pre-S2+S, pre-S2+S, or S.
  • HBV is one of the few DNA viruses that utilize reverse transcriptase in the replication process which involves multiple stages including entry, uncoating, and transport of the virus genome to the nucleus.
  • RNA intermediate that is then reverse transcribed to produce the DNA viral genome.
  • rcDNA viral genomic relaxed circular DNA
  • cccDNA episomal covalently closed circular DNA
  • pgRNA cytoplasmic viral pregenomic RNA
  • cccDNA is an essential component of the HBV replication cycle and is responsible for the establishment of infection and viral persistence.
  • HBV infection results in the production of two different particles: 1) the infectious HBV virus itself (or Dane particle) which includes a viral capsid assembled from the HBcAg and is covered by an envelope consisting of a lipid membrane with HBV surface antigens, and 2) subviral particles (or SVPs) which contain the small and medium forms of the hepatitis B surface antigen HBsAg which are non-infectious.
  • SVPs subviral particles
  • HBV infected cells also secrete a soluble proteolytic product of the pre-core protein called the HBV e-antigen (HBeAg).
  • HBV e-antigen HBV e-antigen
  • the term “HBV” includes any of the genotypes of HBV (A to J). The complete coding sequence of the reference sequence of the HBV genome may be found in for example, GenBank Accession Nos.
  • GI:21326584 and GI:3582357 Amino acid sequences for the C, X, P, and S proteins can be found, for example at NCBI Accession numbers YP_009173857.1 (C protein); YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P protein); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein).
  • Additional examples of HBV mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.
  • HBV Hepatitis B Virus Strain Data
  • the International Repository for Hepatitis B Virus Strain Data can be accessed at www.hpa- bioinformatics.org.uk/HepSEQ/main.php.
  • the term “HBV,” as used herein, also refers to naturally occurring DNA sequence variations of the HBV genome, i.e., genotypes A-J and variants thereof.
  • genotypes A-J genotypes A-J and variants thereof.
  • a study analyzing over 900 publications was performed and data were extracted from 213 records covering 125 countries (Genes 2018, 9(10), 495; doi.org/10.3390/genes9100495).
  • HBV prevalence, and population data the number of infections with each HBV genotype per country was approximated and the genotype distribution among global chronic HBV infections.
  • hepatitis D virus or “HDV” as used herein is a circular, single stranded RNA virus that ranges from 1,672 (strain dFr45, Genbank accession number AX741144) to 1,697 nucleotides (dFr47, GenBank accession number AX741149).
  • a unique open reading frame encodes the small and large hepatitis delta (sHD and IHD, respectively) antigens by way of an editing step in the hepatocyte nucleus.
  • the genetic diversity of HDV is related to the geographic origin of the isolates and there are at least eight genotypes that are referred to as HDV-1 through HDV-8.
  • HDV-2 (previously labeled HDV-IIa) is found in Japan, Taiwan, and Yakoutia, Russia; HDV-4 (previously labeled HDV-IIb) in Taiwan and Japan; HDV-3 which causes epidemics of severe and fulminant hepatitis in the Amazonian region (9); and HDV-5, HDV-6, HDV- 7, and HDV-8 in Africa (LeGal et al., Emerg. Infect. Dis. 12:1447-1450, 2006).
  • HDV-associated disease or "HDV- associated disease” is a disease or disorder that is caused by, or associated with HDV infection or replication.
  • HDV-associated disease includes a disease, disorder or condition that would benefit from reduction in HDV gene expression or replication.
  • Non- limiting examples of HDV-associated diseases include, for example, hepatitis B virus infection, acute hepatits B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; hepatocellular carcinoma.
  • the “WHO Guidelines for prevention, care and treatment of persons with chronic Hepatitis B Infection, 2015” is used herein as a source for information and definitions related to hepatitis B virus, hepatitis B infection, and compositions and methods for treatment of hepatitis B infection.
  • Acute HBV infection is used herein as new-onset hepatitis B infection that may or may not be icteric or symptomatic. Diagnosis is based on detection of hepatitis B surface antigen (HBsAg) and IgM antibodies to hepatitis B core antigen (anti-HBc). Recovery is accompanied by clearance of HBsAg with seroconversion to anti-HBs (antibodies to hepatitis B surface antigen), usually within 3 months.
  • “Chronic HBV infection” or “chronic hepatitis B” is used herein as persistence of hepatitis B surface antigen (HBsAg) for six months or more after acute infection with HBV.
  • “Immune-tolerant phase” as used herein is the high replicative phase of infection seen in the early stage of CHB among people infected at birth or in early childhood.
  • “Immune-active phase” as used herein is the phase of hepatitis B e antigen (HBeAg)-positive disease characterized by fluctuating aminotransferases and high HBV DNA concentrations. May result in seroconversion from HBeAg to anti-HBe (antibody to hepatitis B e antigen).
  • “Inactive phase” or “immune control phase” as used herein is the low replicative phase of chronic hepatitis B characterized by HBeAg negativity, anti-HBe positivity, normal alanine aminotransferase (ALT) and HBV DNA concentration below 2000 IU/mL.
  • “HBeAg seroconversion” as used herein is the loss of HBeAg and seroconversion to anti-HBe-positive.
  • “HBeAg-negative chronic hepatitis B (immune escape phase)” as used herein is HBV infection in a subject that is HBeAg-negative, but anti-HBe-positive disease with variable levels of HBV replication and liver injury.
  • HBsAg seroconversion as used herein is understood as the loss of HBsAg and seroconversion to anti-HBs positive.
  • HBeAg reversion as used herein is understood as the reappearance of HBeAg in a person who was previously HBeAg negative, and usually associated with increased HBV replication.
  • Cirrhosis as used herein is an advanced stage of liver disease characterized by extensive hepatic fibrosis, nodularity of the liver, alteration of liver architecture and disrupted hepatic circulation.
  • cirrhosis Decompensated cirrhosis is understood as a clinical complications of cirrhosis become manifest, including jaundice, ascites, spontaneous bacterial peritonitis, oesophageal varices and bleeding, hepatic encephalopathy, sepsis and renal failure
  • HBsAg Hepatitis B surface antigen
  • HBcAg Hepatitis B core antigen
  • Hepatitis B e antigen Viral protein found in the high replicative phase of hepatitis B. HBeAg is usually a marker of high levels of replication with wild type virus, but is not essential for viral replication.
  • Hepatitis B surface antibody as used herein is understood as an antibody that binds specifically to HBsAg. Anti-HBs develop in response to HBV vaccination and during recovery from acute hepatitis B, denoting past infection and immunity.
  • Hepatitis B e antigen antibody or “Anti-HBe”as used herein is understood as an antibody that binds specifically to HBeAg Detected in persons with lower levels of HBV replication but also in HBeAg-negative disease (i.e. HBV that does not express HBeAg).
  • Hepatitis B core antibody as used herein is understood as an antibody to hepatitis B core (capsid) protein. Anti-HBc antibodies are not neutralizing antibodies and are detected in both acute and chronic infection.
  • IgM anti-HBc is a subclass of anti-HBc that is detected in acute hepatitis B, but can be detected by sensitive assays in active chronic HBV.
  • IgG anti-HBc is a subclass of anti-HBc that is detected in past or current infection.
  • Oxccult HBV infection is a persistent infection present in persons who have cleared hepatitis B surface antigen, i.e. they are HBsAg negative but HBV DNA positive, although at very low levels (invariably ⁇ 200 IU/mL). Most are also anti-HBc positive.
  • Treatment failure as used herein includes both primary or secondary treatment failure.
  • Primary antiviral treatment failure may be defined as failure of an antiviral drug to reduce HBV DNA levels by ⁇ 1 x log10 IU/mL within 3 months of initiating therapy. Secondary antiviral treatment failure may be defined as a rebound of HBV DNA levels of ⁇ 1 x log10 IU/mL from the nadir in persons with an initial antiviral treatment effect ( ⁇ 1 x log10 IU/mL decrease in serum HBV DNA).
  • Treatment failure and drug resistance may be suspected based on features including: receiving antiviral drugs with a low barrier to resistance together with documented or suspected poor adherence, laboratory measures such as an increase in serum aminotransferases, or evidence of progressive liver disease.
  • ALT level is an assessment of ALT levels over time. ALT levels fluctuate in persons with chronic hepatitis B and require longitudinal monitoring to determine the trend. Upper limits for normal ALT have been defined as below 30 U/L for men and 19 U/L for women, although local laboratory normal ranges should be applied.
  • Persistently abnormal or normal may be defined as three ALT determinations above or below the upper limit of normal, respectively, made at unspecified intervals during a 6–12-month period or predefined intervals during a 12-month period.
  • the term “nucelot(s)ide analog” or “reverse transcriptase inhibitor” is an inhibitor of DNA replication that is structurally similar to a nucleotide or nucleoside and specifically inhibits replication of the HBV cccDNA and does not significantly inhibit the replication of the host (e.g., human) DNA.
  • Such inhibitors include Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (TAF), Lamivudine, Adefovir dipivoxil, Entecavir (ETV), Telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-Acetyl-Cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380/LB-80380), and tenofvir-exaliades (TLX/CMX157).
  • TDF Tenofovir disoproxil fumarate
  • TAF Tenofovir alafenamide
  • Lamivudine Adefovir dipivoxil
  • Entecavir ETV
  • Telbivudine AGX-1009
  • the nucelot(s)ide analog is Entecavir (ETV).
  • Nucleot(s)ide analogs are commercially available from a number of sources and are used in the methods provided herein according to their label indication (e.g., typically orally administered at a specific dose) or as determined by a skilled practitioner in the treatment of HBV.
  • a “therapeutic HBV vaccine,” and the like can be a peptide vaccine, a DNA vaccine including a vector-based vaccine, or a cell-based vaccine that induces an immune response, preferably an effector T cell induced response, against one or more HBV proteins.
  • the vaccine is a multi-epitope vaccine that is cross-reactive with multiple HBV serotypes, preferably all HBV serotypes.
  • HBV vaccines A number of therapeutic HBV vaccines are known in the art and are at various stages of pre-clinical and clinical development.
  • Protein based vaccines include hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) vaccines (e.g., Li et al., 2015, Vaccine.33:4247-4254, incorporated herein by reference).
  • Exemplary DNA vaccines include HB-110 (Genexine, Kim et al., 2008.
  • Exemplary protein based vaccines include Theravax/DV-601 (Dynavax Technologies Corp.), EPA-44 (Chongqing Jiachen Biotechnology Ltd.), and ABX 203 (ABIVAX S.A.).
  • Exemplary cell based vaccines include HPDCs-T immune therapy (Sun Yat-Sen University).
  • Combination vaccines and products are also known and include HepTcellTM (FP-02.2 vaccine (peptide)+IC310 Adjuvant (a combination peptide-oligonucleotide adjuvant), (see U.S. Patent Publication Nos.2013/0330382, 2012/0276138, and 2015/0216967, the entire contents of each of which is incorporated herein by reference)); GS-4774 (Gilead, a fusion protein S.
  • HBsAg and HBsAg protein+HBcAg and HBsAg in MVA expression vector Backes et al., 2016, Vaccine.34:923-32, and WO2017121791, both of which are incorporated herein by reference).
  • adjuvant is understood to be an agent that promotes (e.g., enhances, accelerates, or prolongs) an immune response to an antigen with which it is administered to elicit long-term protective immunity. No substantial immune response is directed at the adjuvant itself.
  • adjuvants include, but are not limited to, pathogen components, particulate adjuvants, and combination adjuvants (see, e.g., www.niaid.nih.gov/research/vaccine-adjuvants-types).
  • Pathogen components e.g., monophosphoryl lipid A (MPL), poly(I:C), polyICLC adjuvant, CpG DNA, c-di-AMP, c-di- GMP, c-di-CMP; short, blunt-ended 5 ⁇ -triphosphate dsRNA (3pRNA) RIG-1 ligand, and emulsions such as poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP)) can help trigger early non-specific, or innate, immune responses to vaccines by targeting various receptors inside or on the surface of innate immune cells.
  • the innate immune system influences adaptive immune responses, which provide long-lasting protection against the pathogen that the vaccine targets.
  • Particulate adjuvants form very small particles that can stimulate the immune system and also may enhance delivery of antigen to immune cells.
  • Combination adjuvants e.g., AS02, AS03, and AS04 (all GSK); MF59 (Novartis); ISCOMATRIX® (CSL Limited); and IC31® (Altimmune) elicit multiple protective immune responses.
  • Adjuvants that have a modest effect when used alone may induce a more potent immune response when used together.
  • adjuvants promote a humoral as well as a cellular immune response.
  • Such adjuvants include, for example, polyI:C adjuvant, a polyICLC adjuvant, a CpG adjuvant, a STING agonist (a c-di- AMP adjuvant, a c-di-GMP adjuvant, or a c-di-CMP adjuvant), an ISCOMATRIX® adjuvant, a PCEP adjuvant, and a Rig-I-ligand adjuvant.
  • Immune stimulators include, but are not limited to, pegylated interferon alfa 2a (PEG-IFN-alpha-2a), interferon alfa-2b, PEG-IFN-alpha-2b, interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor (e.g., Myrcludex), an oligonucleotide that inhibits the secretion or release of HBsAg (e.g., REP 9AC), a capsid inhibitor (e.g., Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g., IHVR-25).
  • PEG-IFN-alpha-2a pegylated interferon alfa 2a
  • interferon alfa-2b PEG-IFN-alpha-2b
  • interferon lambda a
  • an immune stimulator can include a viral capsid, optionally an empty viral capsid, e.g., MVA capsid.
  • Immune stimulators can also include immune checkpoint regulators. Immune checkpoint regulators can be stimulatory or inhibitory. As used herein, immune checkpoint regulators potentiate an immune response Immune checkpoint regulators include, but are not limited to, CTLA-4 inhibitors, such as ipilimumab, PD-1 inhibitors, such as Nivolumab, Pembrolizumab, and the BGB-A317 antibody.
  • PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab, in addition to an affimer biotherapeutic.
  • an HBV-associated disease is chronic hepatitis B (CHB).
  • CHB chronic hepatitis B
  • subjects have been infected with HBV for at least five years.
  • subjects have been infected with HBV for at least ten years.
  • subjects became infected with HBV at birth.
  • an HBV-associated disease is HDV.
  • Subjects having chronic hepatitis B disease are immune tolerant, have an active chronic infection accompanied by necroinflammatory liver disease, have increased hepatocyte turn-over in the absence of detectable necroinflammation, or have an inactive chronic infection without any evidence of active disease, and they are also asymptomatic.
  • Subjects having chronic hepatitis B disease are HBsAg positive and have either high viremia (>10e4 HBV-DNA copies/ml blood) or low viremia ( ⁇ 10e3 HBV-DNA copies/ml blood). Patients with chronic active hepatitis, especially during the high replicative state, may have symptoms similar to those of acute hepatitis.
  • the persistence of HBV infection in CHB subjects is the result of ccc HBV DNA persistence.
  • a subject having CHB is HBeAg positive.
  • a subject having CHB is HBeAg negative.
  • the decrease can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.
  • a log 10 scale is typically used to describe the level of antigenemia (e.g., HBsAg level in serum) or viremia (HBV DNA level in serum). It is understood that a 1 log 10 decrease is a 90% decrease (10% remaining), a 2 log 10 decrease is a 99% decrease (1% remaining), etc.
  • a disease marker is lowered to below the level of detection.
  • the expression of a disease marker is normalized, i.e., decreased to a level accepted as within the range of normal for an individual without such disorder, e.g., the level of a disease marker, such as, ALT or AST, is decreased to a level, preferably persistently decreased to a level, accepted as within the range of normal for an individual without such disorder.
  • the disease associated level is elevated from the normal level, the change is calculated from the upper level of normal (ULN).
  • the disease associated level is decreased from the normal level, the change is calculated from the lower level of normal (LLN).
  • the lowering is the percent difference in the change between the subject value and the normal value.
  • a normal AST level can be reported as 10 to 40 units per liter.
  • a subject has an AST level of 200 units per liter (i.e., 5 times the ULN, 160 units per liter above the upper level of normal) and, after treatment, the subject has an AST level of 120 units per liter (i.e., 3 times the ULN, 80 units per liter above the upper level of normal)
  • the elevated AST would be lowered towards normal by 50% (80/160).
  • Persistently abnormal or normal may be defined as three AST determinations above or below the upper limit of normal, respectively, made at unspecified intervals during a 6–12-month period or predefined intervals during a 12-month period.
  • the expression of the HBV gene may be assessed based on the level, or the change in the level, of any variable associated with HBV gene expression, e.g., an HBV mRNA level, an HBV protein level, or an HBV cccDNA level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject, e.g., levels may be monitored in serum.
  • a sample derived from a subject e.g., levels may be monitored in serum.
  • only one dose of the guide RNA or composition provided herein is adminietered to a subject with HBV infection to treat HBV infection.
  • more than one dose (e.g., 2, 3, 4, or 5 doses) of the guide RNA or composition provided herein are adminietered, and each dose may be separated by 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, or 24 weeks.
  • two doses of the guide RNA or composition provided herein is adminietered to a subject with HBV infection, and the second dose is administered after about 1 month, 3 month, or 6 month after the first dose.
  • the methods provided herein comprises (1) administering a first dose of the HBV-targeting guide RNA provided herein and an RNA- guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or a composition provided herein to a subject with HBV infection, (2) determining a clinical marker associated with HBV infection (e.g., serum HBV DNA or liver HBV ccc DNA, serum or liver HBV antigen, e.g., HBsAg or HBeAg), and (3) administering a second dose of the agent if the clinical marker indicates the presence of HBV infection (e.g., if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000
  • the clinical marker associated with HBV infection is measured after a period of time after the first dose (e.g., 1 month, 3 months, or 6 months after the first dose).
  • the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose.
  • step (2) and step (3) can be repeated multiple rounds until the clinical marker indicates the absence or low HBV infection, for example e.g., if the HBsAg protein level or the HBcrAg protein level is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml).
  • Genomic DNA was extracted using QuickExtractTM DNA Extraction Solution (Lucigen, Cat. QE09050) according to the manufacturer's protocol. To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed around the target site within the gene of interest and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field. [0460] Additional PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument.
  • Illumina manufacturer's protocols
  • the reads were aligned to either the human reference genome (e.g., hg38) or HBV genotype D sequence contained in HepAD38 after eliminating those having low quality scores. Reads that overlapped the target region of interest were re-aligned to the local genome sequence to improve the alignment. Then the number of wild type reads versus the number of reads which contain C-to-T mutations, C-to-A/G mutations or indels was calculated. Insertions and deletions were scored in a 20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the 20 bp scoring region divided by the total number of sequencing reads, including wild type.
  • Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the 20 bp scoring region divided by the total number of sequencing reads, including wild type.
  • C-to-T mutations or C-to-A/G mutations were scored in a 40 bp region including 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence.
  • the C-to-T editing percentage is defined as the total number of sequencing reads with either one or more C-to-T mutations within the 40 bp region divided by the total number of sequencing reads, including wild type. The percentage of C-to-A/G mutations are calculated similarly.
  • lipid nanoparticles Preparation of lipid nanoparticles [0462]
  • the lipid nanoparticle (LNP) components were dissolved in 100% ethanol at various molar ratios.
  • the RNA cargos e.g., Cas9 mRNA and sgRNA
  • the RNA cargos were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.
  • the LNPs used contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), also called herein Lipid A, cholesterol, distearoylphosphatidylcholine (DSPC), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat.
  • the LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6.
  • the LNPs used comprise a single RNA species such as Cas9 mRNA or a sgRNA.
  • LNP are similarly prepared with a mixture of Cas9 mRNA and a guide RNA.
  • the LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solution and one volume of water.
  • the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. Then, a fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840 FIG.2).
  • the LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting mixture was then filtered using a 0.2 ⁇ m sterile filter.
  • Example 1.3 In vitro transcription (“IVT”) of mRNA [0464]
  • IVTT In vitro transcription
  • Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase.
  • the linearized plasmid DNA containing a T7 promoter, and a sequence for transcription was linearized by restriction endonuclease digestion followed by heat inactivation of the reaction mixture and purified from enzyme and buffer salts.
  • Messenger RNA was synthesized and purified using standard techniques known in the art.
  • Streptococcus pyogenes (“Spy”) Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs:706 or 712 (see sequences in Table 40).
  • SEQ ID NOs: 706, 712, or other SEQ ID NOs encoding open reading frames are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above).
  • Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NO: 701 in Table 40.
  • Neisseria meningitidis (Nme) Cas 9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 710 or 715 (see sequences in Table 40).
  • SEQ ID NO: 710, 715, or other SEQ ID NOs encoding open reading frames are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above).
  • Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NOs: 703 and 704 in Table 40.
  • Uracil glycosylase inhibitor was generated from plasmid DNA encoding an open reading frame according to SEQ ID NO: 708.
  • SEQ ID NO: 708, or other SEQ ID NOs encoding open reading frames are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above).
  • Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NO: 702 in Table 40.
  • Guide RNAs are chemically synthesized by methods known in the art. Example 1.4.
  • HepAD38 Preparation for Transfection and Experiment Timeline
  • HepAD38 (Ladner et al.1997 Antimicrobial Agents and Chemotherapy 41(8): 1715-1720), a human liver cell line licensed from Fox Chase Cancer Center, contains an integrated, tetracycline-responsive, CMV-IE-promoter-driven copy of HBV Genotype D, ayw strain.
  • DMEM/F121:1 media (Corning 10-092-CM) containing 10% Tet-safe fetal bovine serum (Gibco, A4736401), 1X non-essential amino acids (Corning, 25-025-C1), 1 mM Sodium Pyruvate (Corning 25-000-Cl), and 1X Penicillin-Streptomycin (Gibco 15070-063).
  • Complete media was supplemented with 1 mg/mL Doxycycline (Sigma-Aldrich, D9891) to suppress inducible surface antigen production and 250 ⁇ g/mL G418 Sulfate selection marker (Corning, 30-234- CR) to eliminate cells that did not carry the integrated copy of HBV.
  • HepAD38 cells were thawed and passaged in complete media with supplements in a 10 cm collagen coated dish. Prior to plating cells for transfection, complete media without supplements was prepared and cells were plated in 96-well collagen coated plates at a density of 20,000 cells/well. Supplements were removed prior to transfection to induce surface antigen production and remove elements that may interfere with the transfection or cell health.
  • a typical experimental timeline for HepAD38 experiments was as follows. Cells were plated as described above on Day 0. Transfection was performed 24 hours later on Day 1. On Day 4, upon completion of editing, transfection media was removed from the plate and replaced with fresh complete media without supplements. The cells were maintained for another 7 days to allow for surface antigen accumulation in cell culture media. On Day 11, cell culture media was collected and stored at -80°C for downstream assays. Cells were lysed in 50 ⁇ L/well QuickExtractTM DNA Extraction Solution (Lucigen, QE09050) and stored at - 80°C for downstream assays.
  • HepAD38 cells carry the Hepatitis B virus integrated into the genome. To harvest viral particles, 3 million HepAD38 cells were plated in a collagen-coated 10 cm dish in complete cell media described in Example 1.4 (without supplements). After seven days, media was harvested and replaced with fresh media (cells are not split). After seven days, media was harvested again. Harvested media was stored at -80 ⁇ . ⁇ [0472] To precipitate viral particles, harvested media was centrifuged for 10 min at 1,500 x g to pellet cell debris.
  • the supernatant was moved to a new tube and one volume of PEG-itTM Virus Precipitation Solution (System Biosciences, LV810A-1) was added to 4 volumes of supernatant.
  • the tube was inverted several times and stored at 4 ⁇ overnight. The following day, the mixture was centrifuged at 1,500 x g for 30 min at 4 ⁇ to pellet the viral precipitate.
  • the supernatant was aspirated and the pellet was resuspended in 1/10 of the original volume in complete media. The resulting viral inoculum was aliquoted and stored at -80 ⁇ .
  • HBsAg MSD ELISA Protocol This MSD assay was used to determine HBsAg concentrations in either cell culture media samples or in mouse serum samples.
  • Capture antibody (Monoclonal Anti-HBV antigen HBsAg antibody produced in mouse, Sigma, SAB4700767) and detector antibody (Anti-Hepatitis B Virus Surface Antigen antibody produced in mouse, Abcam, ab8256) were buffer exchanged to PBS using Amicon Centrifugal Unit (Sigma, UFC5050) prior to use.
  • Biotinylation One tube containing the biotinylation labeling reagent (Thermo Fisher, A39259) was thawed at 4 ⁇ . A 2 nmol/ ⁇ L capture solution was prepared by mixing 6.7 ⁇ l of biotin solution into 100 ⁇ l 1 mg/ml buffer exchanged capture antibody.
  • Capture solution was incubated at room temperature for 2 hours in the dark, then was kept at 4 ⁇ overnight.
  • Amicon centrifugal unit was used to remove residual biotin.
  • Sulfo-tag conjugation A 3 nmol/ ⁇ L sulfo-tag (Meso Scale Discovery, R91AO-1) solution was prepared in cold dH2O.13.4 ⁇ L of the sulfo-tag solution was added into 100 ⁇ L of 1 mg/mL buffer exchanged detector antibody. Detector solution was incubated at room temperature in the dark for 2 hours, then was kept at 4 ⁇ overnight. Amicon centrifugal unit was used to remove residual sulfo-tag.
  • Blocker B Buffer 1% and 0.5% Blocker B Buffer (Sigma, RPN2125V) solutions were prepared in PBST. A 2 ⁇ g/mL solution of capture antibody was prepared in 0.5% Blocker B buffer.25 ⁇ L/well of the capture solution was used to coat the plate (Meso Scale Discovery, L15SA-1). The sealed plate was shaken at 700 rpm at room temperature for 1 hour. [0479] Capture solution was removed and 150 ⁇ L of 1% Blocker B buffer was added per well. Sealed plate was shaken at 700 rpm at room temperature for 30 min. Plate was washed 3x with 200 ⁇ L/well PBST.
  • Standard curve was prepared in 0.5% Blocker B buffer using (CellBioLabs- Recombinant HBsAg Standard, 50043C). The standard ranged from 500 ng/mL to 0 ng/mL using a 5-fold serial dilution scheme.
  • In vitro samples were diluted 1:5 in 0.5% Blocker B buffer.50 ⁇ L/well of either standard or sample was added to the plate (standards were run in duplicate). The sealed plate was shaken at 700 rpm at room temperature for 1 hour. Plate was washed 3x with 200 ⁇ L/well PBST.
  • Example 1.7 Immunofluorescence Assay for Intracellular HBsAg Detection Via Microscopy
  • HepAD38 cells were prepared as in Example 1.4, fixed and stained for intracellular HBsAg detection via microscopy post transfection.
  • HBsAg in cells treated with a S-Antigen knockdown guide were evaluated against untreated cells by high content microscopy (CX7 LZR, ThermoFisher).
  • CX7 LZR ThermoFisher.
  • Cells were rinsed with PBS and then fixed using a 4% paraformaldehyde solution in PBS and incubated at room temperature for fifteen minutes. Cells were washed twice with PBS. to permeabilize the cells a 0.5% TritonX100 solution in PBS was prepared and incubated at room temperature on the cells for twenty minutes. Cells were washed twice with 100uL PBS. Blocking was performed using DAKO Blocking Buffer (DAKO, X090930- 2) with slow rocking at room temperature for thirty minutes.
  • DAKO Blocking Buffer DAKO, X090930- 2
  • the primary antibody a Mouse anti-HBsAg antibody (Sigma, SAB4700767), was diluted 1:400 in DAKO Antibody Dilution Buffer (DAKO, K800621-2).
  • DAKO DAKO Antibody Dilution Buffer
  • the blocking buffer was removed from the plate and 50 ⁇ L of the primary antibody solution was added and allowed to incubate with rocking overnight at 4 ⁇ . The following day, the plate was washed twice with 0.05% Tween20 in PBS, then washed twice with PBS.
  • a secondary antibody solution was prepared by diluting a Goat anti- Mouse Alexa FluorTM Plus 488 antibody 1:500 (ThermoFisher Scientific, A32723) and Hoechst stain 1:10,000 (ThermoFisher Scientific, 33342) in Antibody Dilution Buffer. Secondary antibody/Hoechst solution was added to the plate and incubated for about two hours at room temperature with slow shaking. The plate was washed twice with 0.05% Tween20 in PBS and allowed to incubate with the second Tween20 wash for fifteen minutes at room temperature. PBS was added to the plate and imaged and analyzed using a CX7 high- content microscope with SpotDetector Bioassay for intracellular HBsAg detection.
  • Example 2 Example 2
  • Nme2Cas9 meningitidis
  • Nme2-BC22n an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 715), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), and each gRNA from a set of Nme2 gRNAs targeting the integrated HBV genome (Table 17) were transfected.
  • C-to-T editing with Spy and Nme2 was compared to indel formation with their respective cleavases, either an mRNA encoding Spy Cas9 (open reading frame SEQ ID NO: 712) or an mRNA encoding Nme2 Cas9 (open reading frame SEQ ID NO: 710).
  • Spy sgRNAs included chemical modifications shown in Table 12.
  • Nme sgRNAs included the chemical modifications shown in Table 14.
  • Table 17 List of guide RNAs tested Example 2.1.
  • Cell preparation and Transfection [0487] HepAD38 cells were thawed, passaged and transfected as described in Example 1.4 with lipoplexes formed with UGI, a gRNA targeting the HBV surface antigen in the integrated HBV genome of HepAD38, and the appropriate nuclease Spy-BC22n or Nme2-BC22n for the guide RNA.
  • Cleavase lipoplexes also contained cleavase mRNA, UGI mRNA, and guide at the same RNA ratios.
  • Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Lipofection reagent was combined with a cargo by mixing each lipid-formulated RNA species at a 1:1:1 weight ratio: effector mRNA (base editor or cleavase): UGI mRNA: gRNA. The materials were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6.
  • Table 18 and Figure 1 show the NGS sequencing and predicted phenotypic outcomes for all guides targeting the HBV S-Antigen with either Spy-BC22n or Spy-cleavase.
  • Table 19 and Figure 2 show the NGS sequencing and predicted phenotypic outcomes for all guides targeting the HBV S-Antigen with Nme2-BC22n and Nme2-cleavase.
  • Table 18- Genome editing and predicted phenotypic outcomes for guides targeting HBV S-Antigen with Spy-BC22n and Spy-cleavase Table 19- Genome editing and predicted phenotypic outcomes for guides targeting HBV S-Antigen with Nme2-BC22n and Nme2-cleavase Example 3.
  • HepAD38 cells were transfected with gRNA dose-response curve to determine the relative potency, maximum C-to-T editing and maximum predicted knockout level of gRNA targeting the integrated S-antigen locus of HepAD38.
  • Example 3.1 Cell preparation and transfection
  • HepAD38 cells were thawed, passaged and transfected as described in Example 1.4 with a combination of an mRNA encoding Spy Cas9 base editor (SEQ ID NO:706), an mRNA encoding UGI (SEQ ID NO: 708) and a Spy gRNA targeting the integrated S-antigen locus of HepAD38, or a combination of an mRNA encoding Nme2 Cas9 base editor (SEQ ID NO: 715), an mRNA encoding UGI (SEQ ID NO: 708), and a gRNA targeting the integrated S-antigen locus of HepAD38.
  • Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Lipofection reagent was combined by mixing separately each RNA species: base editor mRNA (Spy- or Nme2-BC22n), UGI mRNA, or gRNA. The materials were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The resulting bulk-mixed lipoplex material was pre-incubated with 10% FBS (Corning, 35-101-CV) in complete media (plus Doxycycline) at 37 ⁇ for 10 min before addition to HepAD38 cells.
  • FBS lipid amine to RNA phosphate
  • Example 3.2 Evaluation of C-to-T editing outcomes by next generation sequencing (NGS) [0495] Seventy-two hours post transfection, cells were subjected to lysis, PCR amplification of the HBV S-Antigen locus, and subsequent NGS analysis, as described in Example 1.1.
  • Figures 3A and 3B and Tables 20A and 20B show editing starting at a dose of 100 nM guide using Spy-BC22n or Nme2-BC22n base editor at a constant dose.
  • Figures 4A and 4B and Tables 21A and 21B show editing starting at a dose of 10 nM for both Spy-BC22n and Nme2-BC22n HBV S-antigen guides.
  • HepAD38 cells were thawed, passaged, plated, and transfected as described in Example 1.4.
  • One background control well in each plate was plated with HepG2 (parental) cells at the same density as HepAD38 cells.
  • the HepG2 cells served as a background subtraction control for downstream assays.
  • Two sets of biological duplicates were prepared (four plates total). One set was prepared in clear collagen-coated 96-well plates for editing, qPCR and secreted HBsAg and another set in black-walled collagen-coated 96-well plates for the downstream immunofluorescence assay.
  • Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2.
  • Single-cargo lipofection reagent was prepared by mixing separately each RNA species: base editor mRNA (Spy- or Nme2-BC22n), UGI mRNA, or gRNA. Each well received 100 ng of base editor mRNA, 100 ng of UGI mRNA, and 25 nM of gRNA diluted in complete media. The media-lipofection reagent mixture was incubated at 37 ⁇ for 10 min before addition to the cells. The total volume of all three components was 100 ⁇ L per well. [0501] Media change, media collection, and cell lysis were performed as described in Example 1.4. The set of black-walled plates were prepared for cell fixing and staining for an intracellular HBsAg immunofluorescence assay as described in Example 1.7.
  • Example 1.6 An MSD assay evaluating HBsAg knockdown was performed on collected cell media as described in Example 1.6. Cell lysate was used to quantify intracellular total HBV DNA using a qPCR assay described in Example 1.5. Cell lysates were also prepared for NGS analysis as described in Example 1.1. [0502] Table 22A-22B and Table 23A-23B show the NGS outcomes, percent intracellular HBsAg knockdown, percent secreted HBsAg knockdown, and percent intracellular total HBV DNA knockdown for all guides tested using Spy base editor or Nme2 base editor, respectively. Figure 5A-5B and Figure 6A-6B show knockdown levels for both HBsAg and total intracellular HBV DNA for Nme2 base editor and Spy base editor screens, respectively. Guides with >90% HBsAg knockdown or >90% total HBV DNA knockdown were selected for follow-up experiments.
  • Example 5 In vitro evaluation of Nme2-BC22n /UGI + gRNA LNP potency for knockdown of secreted HBV HBsAg in a primary human hepatocyte HBV infection model
  • Guide RNAs identified by screening in HepAD38 cells as described above were further analyzed for potency in primary human hepatocytes (PHH) using an in vitro infection model. Briefly, PHH were first infected with Hepatitis B inoculum, and after an incubation period, LNPs containing either GFP-mRNA or a combination of Nme2-BC22n mRNA + UGI mRNA + gRNA-targeting HBV were delivered to the cells.
  • PHH were plated in a collagen-coated 96-well plate at a density of 30,000 cells/well. After eighteen hours, plating media was removed and replaced with Primary Hepatocyte Maintenance media consisting of William’s E media and hepatocyte maintenance supplements (Gibco, CM4000), One day later, media was removed and replaced with maintenance media containing 2% DMSO for three hours. Media was removed and replaced with HBV inoculum-containing media consisting of maintenance media, 4% PEG-8000, 2% DMSO, and viral inoculum at an MOI of 50. Plates were then centrifuged at 1000xg for thirty minutes before being returned to the incubator.
  • Primary Hepatocyte Maintenance media consisting of William’s E media and hepatocyte maintenance supplements (Gibco, CM4000)
  • HBV inoculum-containing media consisting of maintenance media, 4% PEG-8000, 2% DMSO, and viral inoculum at an MOI of 50. Plates were then centrifuged at 1000x
  • LNP Transfection Three days post HBV infection, infected PHH cells were transfected with single LNP formulations (formulated as described in Example 1.2) containing either an mRNA encoding GFP (open reading frame SEQ ID NO: 727; negative control) or a combination of gRNA, an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 721), and an mRNA encoding UGI (open reading frame SEQ ID NO: 724) at a 1:2:1 weight ratio.
  • GFP open reading frame SEQ ID NO: 727
  • negative control a combination of gRNA
  • an mRNA encoding Nme2 Cas9 base editor open reading frame SEQ ID NO: 721
  • UGI open reading frame SEQ ID NO: 724
  • the Spy-BC22n and gRNA were packaged in one formulation and UGI mRNA was formulated in a separate formulation.
  • gRNA: BC22n: UGI were delivered at a 1:2:1 weight ratio.
  • Wells received either 11, 33, or 100 ng of total RNA cargo.
  • the LNPs were pre-incubated in maintenance media with 2% DMSO and 6% FBS for 10 minutes before being added to cells. The cells were incubated with the LNPs for four days at which time the media was removed and changed to fresh media with 2% DMSO and 2% FBS. 5.3.
  • HBV S Antigen HBsAg
  • LSBio LS- F37979
  • Media from all conditions was initially diluted at 1:10. However, as a number of samples did not fall within the limit of detection of the assay, the assay was re-run with media at a 1:200 dilution. Bridging samples that were in the linear range from both experiments showed that there was an absolute drop in HBsAg detection on average of 6.1-fold between both ELISA runs. This factor was used to normalize values from both experiments.
  • HBsAg levels were calculated.
  • the average HBsAg levels from HBV gRNA-treated groups was compared to the average HBsAg levels in the GFP-mRNA group to calculate relative percent HBsAg knockdown.
  • Table 24 and Figure 7 show the relative percent HBsAg knockdown of all experimental groups.
  • mice from Jackson Labs were dosed at 6-10 weeks of age with AAV8 and LNP subsequently as indicated, or vehicle (PBS + 0.001% Pluronic for AAV vehicle, TSS for LNP vehicle) via the lateral tail vein.
  • AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein.
  • LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 ⁇ l/gram body weight.
  • blood was collected, and sera was isolated for certain analyses as described further below.
  • HBsAg Human Hepatitis B Virus Surface Antigen
  • HBsAg human Hepatitis B Virus Surface antigen
  • qPCR was run using Absolute qPCR SYBR Green Rox Mix (Thermo Scientific, Cat #AB-1163/A) with a primer set (Fwd-5’-GTTGCCCGTTTGTCCTCTAATTC-3’ (SEQ ID NO: 699), Rev- 5’- GGAGGGATACATAGAGGTTCCTTGA-3’ (SEQ ID NO: 700)) against a highly conserved area of the HBV genome not edited by the tested gRNA.
  • a standard plasmid encoding the same HBV sequence was used to determine DNA copies/mL.
  • sgRNA Seven sgRNA (one control, two Nme2-BC22n, four Spy-BC22n) were assessed for editing via the knock-down of episomal expression of human Hepatitis B Virus Surface antigen (HBsAg) from AAV8-HBV1.2 (SignaGen Laboratories Cat# SL100862) administered as described herein at a dose of 1.2e11 vg/mouse, as measured by ELISA.
  • serum HBsAg expression was assessed for baseline levels via HBsAg ELISA.
  • LNPs tested in this Example were prepared and delivered to mice as described herein one week post the assessment of HBsAg baselines.
  • Two LNPs one containing gRNA and either an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 729) or an mRNA encoding Spy Cas9 base editor (open reading frame SEQ ID NO: 706), and another containing an mRNA encoding UGI (open reading frame SEQ ID NO: 732), were pre-mixed and administered at a dose level of 0.5 mg/kg (with respect to the total RNA cargo content) per body weight of animal.
  • the weight ratio of gRNA:BC22n:UGI was 1:2:0.3.
  • serum HBsAg expression was determined by ELISA at one, two, and four weeks post sgRNA administration.
  • NGS C-to-T %, C-to-A/G % and indel % Example 6.2. Evaluation of Nme2-BC22n LNP in a multidose experiment [0510] Four individual sgRNA (G027224, G030107, G030115, G030120) and one multiplex sgRNA pair (G030107/G030120) were assessed for editing via the knock-down of expression of human Hepatitis B Virus Surface antigen (HBsAg) in a multidose experiment. The human Hepatitis B Virus Surface antigen (HBsAg) ELISA and the total DNA qPCR analysis were performed as described in Example 6.1.
  • HBsAg human Hepatitis B Virus Surface antigen
  • ELISA was performed to measure episomal expression from AAV8-HBV1.2 (SignaGen Laboratories Cat# SL100862) administered as described herein at Day 0 at a dose of 5e10 vg/mouse. At Day 13, serum HBsAg expression was assessed for baseline levels via HBsAg ELISA. Cargos were individually formulated in LNPs, each containing an individual gRNA or mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 729) and another containing an mRNA encoding UGI (open reading frame SEQ ID NO: 732).
  • the LNPs were pre-mixed and administered at a dose level of 0.5 mg/kg (with respect to total RNA cargo content) per body weight of animal at Day 15 and Day 42.
  • the weight ratio of gRNA:BC22n:UGI was 1:2:0.3.
  • Blood was collected at Days 0, 15, 28, 42, and 63, and serum HBsAg expression was determined by ELISA.
  • the animals were euthanized; blood was collected to assess terminal HBsAg and HBV DNA levels in serum.
  • Liver was collected for Next Generation Sequencing (NGS) and editing evaluation at HBV target sites.
  • C-to-T %, C-to-A/G % and indel % were determined by NGS as described in Example 1.1.
  • HBsAg levels were reduced after the first LNP dose and further reduced for all groups after the second dose. At Day 63 raw data and at Day 84 normalized knockdown showed that multi-dosing has a significant benefit to HBsAg knockdown in 4 out of 5 groups.
  • Example 7 - Dose response curve using Nme2 guides [0513] A dose response transfection of guide RNA was performed to compare potency of selected guides from the gRNA screen (Example 4) to previously identified high activity Nme2 guides targeting HBV genotype D that resulted in HBsAg knockdown.
  • HepAD38 cells were thawed, passaged, plated, and transfected as described in Example 1.4 with separate lipoplexes formed individually with an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 715), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), or gRNA targeting the integrated S-antigen, Polymerase, or Epsilon locus of HepAD38.
  • Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2.
  • Single- cargo lipofection reagent was prepared by mixing separately each RNA species: Nme2- BC22n base editor mRNA, UGI mRNA, or gRNA. Each well received 100 ng of base editor and 100 ng of UGI diluted in complete media. A guide RNA dose response curve for each guide tested was formed in complete media with concentrations ranging from 50 nM to 0 nM, titrated using a 3-fold dilution scheme. The media-lipofection reagent mixture was incubated at 37oC for 10 minutes before addition to HepAD38 cells. The total volume of all three components was 100 ⁇ L per well. [0516] Media change, media collection and cell lysis were performed as described in Example 1.4.
  • Table 29 Percent secreted HBsAg (top two doses of gRNA only) for the extended Nme2 lead guide DRC.
  • Nme2-BC22n lead guides were tested in multiplex combinations to evaluate combinatorial effects of a multi-guide treatment relative to a single-guide treatment to NGS outcomes as well as HBsAg and total HBV DNA knockdown.
  • HepAD38 cells were plated in collagen-coated 96-well plates at a density of 20,000 cells/well in complete media (without Doxycycline and G418 Sulfate supplements) as described in Example 1.4.
  • lipid nanoparticles prepared essentially as described in Example 1.2.
  • Lipid nanoparticles used 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG by molarity.
  • the lipid nucleic acid assemblies were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a cargo of 1:2:1 for gRNA:Nme2-BC22 (an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 721)):UGI (mRNA encoding UGI (open reading frame SEQ ID NO: 724)).
  • a dose response curve was formed in complete media with total LNP cargo masses ranging from 80 ng to 0 ng, titrated using a 2-fold dilution scheme.
  • the media-LNP mixture was incubated at 37oC for 10 minutes before addition to HepAD38 cells.
  • a total volume of 100 ⁇ L/well media-LNP mix was delivered.
  • Media change, media collection and cell lysis were performed as described in Example 1.4.
  • An MSD assay evaluating HBsAg knockdown was performed on collected cell media as described in Example 1.6.
  • Cell lysates were also prepared for NGS analysis as described in Example 3.2. NGS data is shown in Table 30. Figures 13 and 14 and Table 31 show percent knockdown of secreted HBsAg measured by MSD assay.
  • Nme2-BC22n lead guides were tested in singleplex and multiplex combinations in a HBV-infected PHH model.
  • Serially diluted LNP were delivered to evaluate combinatorial effects of a multi-guide treatment relative to a single-guide treatment via secreted HBsAg knockdown (MSD) and total intracellular HBV DNA knockdown (ddPCR). 9.1.
  • MSD secreted HBsAg knockdown
  • ddPCR total intracellular HBV DNA knockdown
  • 9.1 Cell culture, infection and LNP delivery
  • Primary Human Hepatocytes (ThermoFisher, HU8345) were thawed and recovered in CHRMs media (Gibco, CM7000).
  • HBV inoculum- containing media consisting of maintenance media, 4% PEG-8000, and viral inoculum, MOI 50 (viral inoculum was prepared according to Example 1.5). Uninfected cells received maintenance media with 4% PEG-8000 only. Plates were centrifuged at 1000g for 30 minutes at 37 degrees Celsius before being moved to an incubator. Twenty-four and forty-eight hours later, media was removed and replaced with maintenance media supplemented with 2% DMSO and 2% FBS.
  • HBV DNA copy number was measured by ddPCR (Bio-Rad). Using primers designed against conserved regions of the HBV genome and the Hs Actin Beta (ACTB) gene, a ddPCR reaction was prepared using a ddPCR Supermix for probes (Bio-Rad, 1863024) and readout on the droplet reader following the manufacturer’s protocol. HBV levels for each sample were analyzed by normalizing HBV copy number/uL to ACTB copy number/uL.
  • Table 33 Percent reduction in HBV DNA copy number with single and multiplexed HBV guides Example 10 – Comparing potent HBV targeting Nme2-BC22n gRNA with different modification patterns in HBV-infected Primary Human Hepatocytes using lipid nanoparticles [0530] Three different Nme2-BC22n lead spacer sequences were tested head-to-head against a different scaffold modification pattern to determine potency differences in HBV- infected PHH. LNP dose-response curves were delivered to evaluate potency via secreted HBsAg knockdown (MSD). [0531] Cell culture, infection, and LNP delivery were performed as in Example 9.1. 10.1.
  • HBsAg knockdown Measurement of secreted HBsAg was performed as described in Example 1.6. To determine absolute levels of HBsAg for each sample level, the background subtracted signal from each well was interpolated using a four-parameter non-linear curve of the standard HBsAg protein. gRNA knockdown IC50s (nM gRNA) were calculated from each HBsAg knockdown curve and the IC50 from one modification pattern was compared to the other to determine statistical significance. No statistical differences in HBsAg knockdown potency were noted between the different modification patterns. [0533] HBsAg concentration (ng/ml) is shown in Table 34 and Figure 17. HBsAg IC50 data is in Table 35 and Figure 18. Table 34: HBsAg concentration (ng/mL) following treatment with guides with different modification patterns
  • AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein.
  • LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 ⁇ l/gram body weight.
  • blood was collected, and sera was isolated for certain analyses as described further below. 11.1.
  • HBsAg Human Hepatitis B Virus Surface Antigen
  • Serum HBsAg levels were quantitated off a standard curve using 4-parameter logistic fit and expressed as ⁇ g/mL of serum.
  • serum was diluted 1:50 and DNA was isolated using the QE lysis protocol (Lucigen, Cat. # QE09050).
  • ddPCR was run using ddPCR Supermix for probes (Bio-Rad, Cat.
  • sgRNA one control targeting SCAP, three targeting HBV
  • HBsAg human Hepatitis B Virus Surface antigen
  • LNPs containing gRNA:Nme2-BC22n:UGI at a ratio of 1:2:0.3 were pre-mixed and administered at a dose level of 2 mg/kg (with respect to the total RNA cargo content) per body weight of animal. Thereafter, serum HBsAg expression was determined by ELISA two weeks post the first LNP administration. On the same day, the animals were again administered their respective LNPs at a dose level of 2 mg/kg. [0537] Six weeks post the first LNP dose, the animals were euthanized; blood was collected to assess terminal HBsAg and HBV DNA levels in serum. Phenotypic measurements for each guide-specific target sites are shown in Table 36 and Table 37.
  • Serum HBsAg log10 reduction time course relative to individual animal baseline is shown in Figure 19.
  • Log10 knockdown of total serum HBV DNA relative to TSS group at week 6 is shown in Figure 20.
  • the average HBsAg log10 knockdown from baseline was >2 log10 for G027224 and G030115. All guides resulted in >2 log of serum HBV DNA knockdown but due to assay sensitivity this translated in a maximum detectable knockdown of 2.2 log10.
  • AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein.
  • LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 ⁇ l/gram body weight.
  • blood was collected, and sera was isolated for certain analyses as described further below. 12.1.
  • Human Hepatitis B Virus Surface Antigen (HBsAg) analysis [0539] For in vivo studies, blood was collected, and the sera was isolated as indicated.
  • HBsAg human Hepatitis B Virus Surface antigen
  • sgRNA one control targeting Scap, three targeting HBV were assessed for editing via the knock-down of episomal expression of human Hepatitis B Virus Surface antigen (HBsAg) from AAV8-HBV1.2 Genotype A (AB246338.1) or AAV-HBV1.2 Genotype C (AB246345.1) (SignaGen Laboratories Cat# SL100865) administered as described herein at a dose of 1e11 vg/mouse, as measured by ELISA.
  • HBsAg human Hepatitis B Virus Surface antigen
  • AAV8-HBV1.2 Genotype A AB246338.1
  • AAV-HBV1.2 Genotype C AB246345.1
  • G027224 is not fully homologous to the HBV genotypes used in this experiment and has a 1 bp mismatch in guide position 13 relative to Genotype A and a 1 bp mismatich at guide position 16 relative to Genotype C.
  • Table 37 Log10 serum HBsAg reduction from baseline with HBV genotype A
  • Table 38 Log10 serum HBsAg reduction from baseline with HBV genotype C
  • Table 39 Endpoint comparisons of log10 serum HBsAg reduction from baseline Table 40: Additional Sequences

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Compositions and methods are provided for modifying a hepatitis B virus genome.

Description

COMPOSITIONS AND METHODS FOR HEPATITIS B VIRUS (HBV) GENOME EDITING RELATED APPLICATIONS [001] This application claims the benefit of priority to U.S. Provisional Patent Application serial number 63/450,307, filed March 6, 2023, which is hereby incorporated by reference in its entirety. SEQUENCE LISTING [002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 26, 2024, is named ILH-02825_SL.xml and is 6,286,863 bytes in size. BACKGROUND [003] Hepatitis B virus (HBV) is an enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation, and is a major risk factor for development of cirrhosis and hepatocellular carcinoma (HCC). The World Health Organization (WHO) estimates that there are 240 million chronically HBV-infected individuals worldwide, largely in low to middle income countries and that about 650,000 people die annually of complications from HBV infection. Although an effective HBV vaccine is available and efforts to vaccinate infants at birth have been effective in reducing incidence and prevalence of HBV infection, such programs do not have a demonstrable effect on death rates for years (WHO Guidelines for prevention, care and treatment of persons with chronic Hepatitis B Infection, 2015 at apps.who.int/iris/bitstream/handle/10665/154590/9789241549059_eng.pdf). [004] A number of therapeutic agents have been developed for the treatment of HBV that effectively reduce the disease burden of HBV infection, but they are not typically curative as they do not fully eliminate all replicative forms of the virus including the covalently closed circular DNA (cccDNA) that resides in the hepatocyte nucleus and becomes a template for viral replication and transcription of viral RNAs. Nucleotide and nucleoside analogs, typically considered to be the gold standard for treatment of chronic HBV infection due to their safety and efficacy, effectively suppress HBV replication, but do not eliminate cccDNA, do not prevent expression of viral proteins, must be dosed chronically, and can result in the development of resistance. Further, treatment with nucleot(s)ide inhibitors does not fully mitigate the risk of the development of hepatocellular carcinoma (HCC), which remains significant (about 7% of those infected with HBV will develop HCC within 5-7 years despite treatment). Interferon-based therapies can result in seroconversion and cure about 10-15% of patients, thereby allowing discontinuation of treatment, but the agents have severe side effects and must be refrigerated for long-term storage, making them less desirable for use in many countries where HBV infection is prevalent. [005] Treatment of chronic HBV infection is further complicated by the ability of HBV to evade or suppress the immune response resulting in persistence of the infection. Some HBV proteins have immune-inhibitory properties, with hepatitis B s antigen (HBsAg) comprising the overwhelming majority of HBV protein in the circulation of HBV-infected subjects. Additionally, while the removal (via seroconversion) of hepatitis B e antigen (HBeAg) or reductions in serum viremia are not correlated with the development of sustained control of HBV infection off treatment, the removal of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-recognized prognostic indicator of antiviral response to treatment that will lead to control of HBV infection off treatment. However, this only occurs in a small fraction of patients receiving immunotherapy. Therefore, it is possible, although rare, for patients to mount a sufficiently robust immune response to suppress or clear an HBV infection resulting in at least a functional cure of the disease. [006] Hepatitis D virus or hepatitis delta virus (HDV) is a human pathogen. However, the virus is defective and depends on obligatory helper functions provided by the hepatitis B virus (HBV) for transmission; indeed, HDV requires a simultaneous infection with HBV (co- infection) or superimposition on a pre-existing HBV infection (superinfection) to become infectious and thrive. In particular, HDV requires the HBV viral envelope containing the surface antigen of hepatitis B. Therefore, an effective treatment of HBV infection would provide an effective treatment of HDV. SUMMARY [007] Provided herein are compositions and methods for modifying the hepatitis B virus (HBV) genome. Methods are also provided for treating a subject infected with HBV. [008] In some aspects, provided herein is a guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 conitguous nucleotides of nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; B. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; C. a targeting sequence comprising a sequence identical to or complementary to at least 17, 18, preferably 19, or 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1; or D. a targeting sequence comprising a sequence identical to or complementary to at least 20, 21, 22, preferably 23, or 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1. [009] In some aspects, provided herein is a guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of: 1. nucleotides 163-466, nucleotides 1- 843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821- 2460 of SEQ ID NO: 1; 2. nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240- 269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373- 392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629- 648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725- 744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884- 1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867- 2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153- 173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; or 3. a target site selected from a target site of any one of HBV number S1-S93 in Table 1A, optionally a target site of any one of HBV number S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 in Table 1A; B. a targeting sequence comprising a sequence identical to or complementary to 17, 18, preferably 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, or to a targeting sequence of S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 provided in Table 1A; C. a targeting sequence selected from the targeting sequence of G028550, G028551, G028552, G028553, G028554, G028555, G030415, G030418,G030423, G030424, G030426, G030428, G030429, G030430, G030431, G030432, G030435, G030436, G030437, G030439, G030446, G030454, G030456, G030457, G030459, G030460, G030464, G030465, G030468, G030469, G030470, G030471, G030474, G030477, G030478, G030481, G030484, G030485, G030487, G030488, G030489, G030490, G030491, G030492, G030493, G030494, G030496, G030497, G030498, or G030500, optionally G028550, G028554, or G028555; or D. a targeting sequence comprising a sequence identical to or complementary to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1- 1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460; or nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250- 269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686- 705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845- 1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073- 2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891- 2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1. [010] In some embodiments, the guide RNAs provided herein further comprise one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1- 8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c.1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 268); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region. [011] In some embodiments, the guide RNA lacks 6 nucleotides in shortened hairpin 1. [012] In some embodiments, the guide RNA lacks 8 nucleotides in shortened hairpin 1. [013] In some embodiments, H-1 and H-3 are deleted. [014] In some embodiments, the guide RNA further comprises a 3’ tail. [015] In some embodiments, the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length. [016] In some embodiments, the guide RNA comprises an upper stem region comprising a modification to any one or more of US1-US12 in the upper stem region. [017] In some embodiments, the guide RNAs described herein comprise a nucleotide sequence selected from the sequences in Table 3A. [018] In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from the modified Spy guide scaffold sequences in Table 4, wherein the modified nucleotide sequence is 3’ of the guide sequence. [019] In some embodiments, the guide RNAs described herein are modified according to the pattern of a nucleotide sequence selected from the modified Spy guide RNA sequences in Table 15. [020] In some embodiments, the guide comprises a nucleotide sequence selected from the unmodified Spy guide RNA Sequences in Table 3B, wherein the N20’s are collectively a targeting sequence described herein. [021] In some embodiments, each nucleotide of the unmodified Spy guide RNA Sequences in Table 3B is any natural or non-natural nucleotide. [022] In some embodiments, the guide RNA is modified according to a pattern selected from the modification patterns in Table 15, wherein the (mN*)3N17 refers to the targeting sequence described herein in which the first three nucleotides comprises a 2’-O-Me modification and a phosphorothioate linkage. [023] In some embodiments, the guide RNAs described herein comprise a sequence or modification pattern selected from SEQ ID NOs: 269, 273, 274, 362, 366, or 367 as set forth in Table 12. [024] In some aspects, provided is a guide RNA (gRNA) comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of: 1. nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; 2. nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958- 2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330- 353, 755-778, or 713-736 of SEQ ID NO: 1; or 3. a target site selected from any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; B. the guide region comprises a nucleic acid sequence identical to or complementary to 21, 22, preferably 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; or of a targeting sequence of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; C. the guide region is selected from targeting sequence of G027224, G027225, G027228, G030107, G030108, G030115, G030120, G030123, G030125, G030130, G030134, G030138, G030158, G030162, G030167, G030180, or G030181; optionally G027224, G027225, G027228, G030107, G030115, G030120, G030123, G030125, G030130, G030162, or G030167; optionally G027224, G030107, G030115, or G030120; or D. the guide region comprising a sequence identical to or complementary to 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1; or nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740- 763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1. [025] In some embodiments, the conserved region comprises one or more of: (a) a shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region lacks 2-24 nucleotides relative to SEQ ID NO: 455, wherein (i) one or more of nucleotides 37-48 and 53-64 is deleted relative to SEQ ID NO: 455 and optionally one or more of nucleotides 37-64 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2- 10 nucleotides, optionally 2-8 nucleotides relative to SEQ ID NO: 455 wherein (i) one or more of nucleotides 82-86 and 91-95 is deleted relative to SEQ ID NO: 455 and optionally one or more of positions 82-96 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18 nucleotides, optionally 2-16 nucleotides relative to SEQ ID NO: 455, wherein (i) one or more of nucleotides 113-121 and 126-134 is deleted relative to SEQ ID NO: 455 and optionally one or more of nucleotides 113-134 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; wherein one or both nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 455; optionally, wherein at least 10 nucleotides are modified nucleotides. [026] In some embodiments, the conserved region comprises a nucleotide sequence selected from Table 5. [027] In some embodiments, the guide RNA comprises at least one end modification. [028] In some embodiments, the modification comprises a 5’ end modification. [029] In some embodiments, the modification comprises a 3’ end modification. [030] In some embodiments, the guide RNA comprises a modification in a hairpin region. [031] In some embodiments, the modification in a hairpin region is also an end modification. [032] In some embodiments, the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide. [033] In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. [034] In some embodiments, the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide. [035] In some embodiments, the modification comprises a 2’-fluor (2’F) modified nucleotide. [036] In some embodiments, the 5’ end modification comprises a 2’-O-methyl (2’-O- Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide at nucleotides 1-3 of the 5’ end of the guide sequence. [037] In some embodiments, the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motifs in Table 6, and wherein the conserved region is 3’ of the guide region. [038] In some embodiments, the guide RNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 226-234, and wherein the N’s represent the guide sequence of any one of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37. [039] In some embodiments, each nucleotide is any natural or non-natural nucleotide. [040] In some embodiments, the guide RNA is modified according to a pattern selected from SEQ ID NOs: 670-678, wherein the N’s are collectively the guide sequence described herein, wherein N, A, C, G, and U are ribonucleotides (2’-OH), wherein “m” indicates a 2’-O- Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides. [041] In some aspects, provided herein is a composition comprising a guide RNA described herein. [042] In some aspects, provided herein is a composition comprising a combination of guide RNAs comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of target sites selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N37; N24 and N79; N24, N32, and N79; N24, N32, and N37; and N24, N32, and N40; optionally N24 and N37; N7 and N32; N7 and N37; and B. the conserved region comprises a conserved region described herein. [043] In some embodiments, the composition further comprises an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. [044] In some embodiments, the nucleic acid encoding the RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding the RNA-guided DNA binding agent. [045] In some embodiments, the RNA-guided DNA binding agent is a nuclease . [046] In some embodiments, the RNA-guided DNA binding agent is a Cas9 nuclease. [047] In some embodiments, the Cas9 is S. pyogenes Cas9. [048] In some embodiments, the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 707 and 713 or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to a sequence selected from SEQ ID NOs: 707 and 713. [049] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706 and 712. [050] In some embodiments, the Cas9 is Nme Cas9. [051] In some embodiments, Nme Cas9 comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 711, 716, 719, 722, or 730 or an ORF encoding an Nme Cas9 having at least 90% identity to a sequence selected from 600-620. [052] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to a sequence selected from SEQ ID NOs: 705, 710, 715, 718, 721, or 729. [053] In some embodiments, the nuclease has double stranded endonuclease activity. [054] In some embodiments, the nuclease has nickase activity. [055] In some embodiments, the nuclease is catalytically inactive. [056] In some embodiments, the nuclease further comprises a heterologous functional domain. [057] In some embodiments, the nuclease is a nickase and the heterologous functional domain is a deaminase. [058] In some embodiments, the deaminase is a cytidine deaminase or an adenine deaminase. [059] In some embodiments, the deaminase is a cytidine deaminase. [060] In some embodiments, the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. [061] In some embodiments, the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs.707, 716, 719, 722, or 730 or an ORF encoding an amino acid sequence having at least 90% identity to SEQ ID NOs: 707, 716, 719, 722, or 730. [062] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706, 715, 718, 721, or 729. [063] In some embodiments, the composition described herein further comprises a uracil glycosylase inhibitor (UGI) or nucleic acid encoding a UGI, wherein the nuclease polypeptide does not comprise a UGI or the nucleic acid encoding the polypeptide does not encode a UGI. [064] In some embodiments, the UGI comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 709, 725, or 733 or an ORF encoding an amino acid sequence having at least 90% identity to a sequence selected from 709, 725, or 733. [065] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NO: 708, 724, 732, optionally SEQ ID NO: 708. [066] In some embodiments, the ORF is a modified ORF. [067] In some embodiments, the composition described herein further comprises a pharmaceutical excipient. [068] In some embodiments, the guide RNA is associated with a lipid nanoparticle (LNP). [069] In some embodiments, the LNP comprises a cationic lipid. [070] In some embodiments, the cationic lipid is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. [071] In some embodiments, the LNP comprises a helper lipid. [072] In some embodiments, the helper lipid is cholesterol. [073] In some embodiments, the LNP comprises a neutral lipid. [074] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC). [075] In some embodiments, the LNP comprises a stealth lipid. [076] In some embodiments, the stealth lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol 2000 (PEG2k-DMG). [077] In some embodiments, the LNP comprises (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG. [078] In some aspects, provided herein is a pharmaceutical composition comprising the guide RNA described herein or the composition described herein. [079] In some aspects, provided herein is a pharmaceutical composition comprising, or use of, the guide RNA described herein or the composition described herein for inducing a double stranded break or a single stranded break within an HBV gene in a cell, modifying the nucleic acid sequence of an HBV gene in a cell, or reducing expression of an HBV gene in a cell. [080] In some embodiments, the HBV gene is the S gene. [081] In some embodiments, the pharmaceutical composition or use described herein is for reducing expression of the HBV gene in a cell or subject. [082] In some aspects, provided herein is a pharmaceutical composition comprising, or use of, the guide RNA described herein or the composition described herein for treating a subject having hepatitis B. [083] In some embodiments, the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the pharmaceutical composition. [084] In some embodiments, the pharmaceutical composition or use described herein is for use in combination with an immune stimulator. [085] In some embodiments, the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha-2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1 inhibitor, atezolizumab, avelumab, durvalumab, or an affimer biotherapeutic. [086] In some embodiments, the immune stimulator is a vaccine. [087] In some embodiments, the vaccine is a nucleic acid vaccine or a protein-based vaccine. [088] In some embodiments, the nucleic acid vaccine is an RNA vaccine. [089] In some embodiments, the subject has chronic HBV. [090] In some embodiments, the subject is HBe antigen positive. [091] In some embodiments, the subject is HBe antigen negative. [092] In some embodiments, the subject has hepatitis D. [093] In some embodiments, the pharmaceutical composition or use described herein is for treating hepatitis D. [094] In some aspects, provided herein is a method or inducing a double stranded break or a single stranded break within an HBV gene in a cell or reducing expression of an HBV protein in a cell comprising contacting the cell with the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein. [095] In some embodiments, the HBV protein is HBsAg protein or HBcrAg protein. [096] In some embodiments, the cell is in a subject. [097] In some embodiments, the HBsAg protein level or the HBcrAg protein level is measured in a subject sample selected from blood or serum. [098] In some embodiments, the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml. [099] In some embodiments, the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition. [0100] In some embodiments, the method described herein further comprise administration of an immune stimulator. [0101] In some embodiments, the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha-2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1 inhibitor, atezolizumab, avelumab, durvalumab, or an affimer biotherapeutic. [0102] In some embodiments, the immune stimulator is a vaccine. [0103] In some embodiments, the vaccine is a nucleic acid vaccine or a protein-based vaccine. [0104] In some embodiments, the nucleic acid vaccine is an RNA vaccine. [0105] In some embodiments, the subject has chronic HBV. [0106] In some embodiments, the subject is HBe antigen positive. [0107] In some embodiments, the subject is HBe antigen negative. [0108] In some embodiments, the subject has hepatitis D. [0109] In some embodiments, the methods described herein further comprise treating hepatitis D in the subject. [0110] In some embodiments, the method comprises administering one or more doses of the guide RNA and the RNA-guided DNA binding agent, of the nucleic acid encoding an RNA- guided DNA binding agent, or of the composition to a subject, optionally comprising administering two, three, four, or five doses. [0111] In some embodiments, the method described herein comprises administering two or more of the doses, wherein at least two consecutive doses are administered at times separated by at least or about 1 month, 3 months, or 6 months. [0112] In some embodiments, the method described herein comprises, after administering a first dose, determining HBsAg protein level or HBcrAg protein level in blood or serum of the subject; and subsequently administering to the subject a second dose if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml. [0113] In some embodiments, the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose. [0114] In some aspects, use of the guide RNA described herein or the composition described herein in the preparation of a medicament for practicing any of the methods described herein is provided. [0115] In some aspects, provided herein is a liver cell comprising an HBV genome, wherein the HBV genome comprises a sequence having at least 80%, 85%, optionally 90% or 95% identity, optionally 100% identity to at least 20 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356- 375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621- 640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695- 714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982- 3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; wherein the HBV genome comprises a genetic modification in the HBV genome within the sequence having at least 80%, 85%, optionally 90%, or 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245- 264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423- 442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635- 654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1. [0116] In some embodiments, the genetic modification comprises a mutation that generates a stop codon or disrupts initiation of transcription. [0117] In some embodiments, the genetic modification comprises an indel. [0118] In some embodiments, the HBV genome comprises a sequence having at least 90%, 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356- 375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621- 640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695- 714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982- 3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1. [0119] In some embodiments, the liver cell comprises a second genetic modification in the HBV genome within the sequence having at least 80%, 85%, optionally 90% or 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1. [0120] In some embodiments, the genetic modification is within the HBV genome within the sequence having at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity identity to at least 20 contiguous nucleotides of targeting sites in Table 1A, optionally a targeting site of a gRNA with an HBV number selected from S1-S6, S8, S11, S16, S17, S19, S21-25, S28- S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80- S87, S80, S90-S91, and S93, optionally S1, S5, or S6. [0121] In some embodiments, the HBV genome comprises a sequence having 100% identity or at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447- 470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1. [0122] In some embodiments, the HBV genome comprises a sequence having at least 90% or 95% identity; or 100% identity to at least 24 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846 , or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951- 2974, or 2958-2981 , optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1. [0123] In some embodiments, the genetic modification is within the sequence having at least 80%, 85%, optionally 90%, or 95% identity or 100% identity to at least 24 contiguous nucleotides of the target sites in Table 2A, optionally a targeting site of a gRNA with an HBV number selected from N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37; or a targeting site of gRNAs with a combination of HBV numbers selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N37; N24 and N79; N24, N32, and N79; N24, N32, and N37; and N24, N32, and N40; optionally N24 and N37; N7 and N32; N7 and N37. [0124] In some embodiments, the HBV genome is integrated into the liver cell genome. [0125] In some embodiments, the HBV genome is cccDNA. [0126] In some aspects, provided herein a method of modifying a nucleic acid sequence of the HBV genome, the method comprising contacting an HBV-infected liver cell the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein. [0127] In some embodiments, the method is performed in vivo, in vitro, or ex vivo. [0128] In some embodiments, the cell is in a subject infected with HBV. [0129] In some aspects, provided herein a method of treating an HBV infection in a subject, the method comprising administering to the subject the guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition described herein. [0130] In some embodiments, the nucleic acid sequence of the HBV genome encodes an HBV protein. [0131] In some embodiments, the HBV protein is HBsAG or ABcrAg protein. [0132] In some embodiments, the method further comprises determining the HBsAg protein level or the HBcrAg protein level in a subject blood or serum sample. [0133] In some embodiments, the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml. [0134] In some embodiments, the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition. [0135] In some embodiments, the methods described herein further comprise administering an immune stimulator to the subject. DETAILED DESCRIPTION [0136] Reference will now be made in detail to certain embodiments of the disclosure, examples of which are illustrated in the accompanying drawings. While the present teachings are described in conjunction with various embodiments, it is not intended to limit the present teachings to those embodiments. On the contrary, the present teaching encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. [0137] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells (e.g., a population of cells) and the like. [0138] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. [0139] The use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). [0140] The term “or” is used in an inclusive sense in the specification, i.e., equivalent to “and/or,” unless the context clearly indicates otherwise. [0141] The term “about,” when used before a list, modifies each member of the list. The term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement. When “about” is present before the first value of a series, it can be understood to modify each value in the series. [0142] Ranges are understood to include the numbers at the end of the range and all logical values therebetween. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%. [0143] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing a upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least,” “up to,” or other similar language modifies a number, it can be understood to modify each number in the series. [0144] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified. [0145] As used herein, ranges include both the upper and lower limit. [0146] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates. [0147] As used herein, “detecting an analyte” and the like is understood as performing an assay in which the analyte can be detected, if present, wherein the analyte is present in an amount above the level of detection of the assay. [0148] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition or 100% encapsulation) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to a level below the level of detection of the assay, and 100% encapsulation is understood as no material intended for encapsulation can be detected outside the vesicles. [0149] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. I. Definitions [0150] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings: [0151] “Polynucleotide” and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy, 2’ halide, or a 2’-O-(2-methoxyethyl) (2’-O-moe) substitutions. An RNA may comprise one or more deoxyribose nucleotides, e.g. as modifications, and similarly a DNA may comprise one or more ribonucleotides. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1- methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No.5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional nucleosides with 2’ methoxy substituents, or polymers containing both conventional nucleosides and one or more nucleoside analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acid includes “unlocked nucleic acid” enables the modulation of the thermodynamic stability and also provide nuclease stability. RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA. [0152] “Polypeptide” as used herein refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like. [0153] “Guide RNA,” “gRNA,” and simply “guide” are used herein interchangeably to refer to, for example, either a single guide RNA or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA strand (as a single guide RNA, sgRNA) or, for example, in two separate RNA strands (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations. [0154] As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. [0155] A “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.” A guide sequence can be about 20 nucleotides in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9), or guide sequence can be about 24 nucleotides in length, e.g., in the case of Neisseria meningitidis (Nme Cas9), and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used. [0156] For example, Spy Cas9 guides can be 16-, 17-, preferably 18-, 19-, or 20- nucleotides in length, such that, in some embodiments, the Spy Cas9 targeting sequence comprises 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, from nucleotides 153- 173, 245-269, 153-172, 154-173, 245-264, 248-267, 250-269, 364-383, 621-665, 694-716, 1176- 1216, 1187-1216, 1383-1418, 1398-1418, 1424-1448, 1701-1724, 2296-2318, 2867-2894, 2901- 2928, 2976-3001 , 621-640, 629-648, 636-655, 694-713, 695-714, 697-716, 796-815, 1191-1210, 1197-1216, 1383-1402, 1398-1417, 1399-1418, 1424-1443, 1429-1448, 1529-1548, 1540-1559, 1569-1588, 1605-1624, 1701-1720, 1705-1724, 1867-1886, 1880-1899, 1883-1902, 2072-2091, 2296-2315, 2299-2318, 2867-2886, 2868-2887, 2875-2894, 2875-2894, 2901-2920, 2902-2921, 2909-2928, 2948-2967, 2956-2975, 2976-2995, 2982-3001, 2982-3001, or 3048-3067, optionally from nucleotides 153-173, 245-269, 153-172, 154-173, 245-264, 248-267, 250-269, or 364-383 of SEQ ID NO: 1, or a reverse complement thereof; for example, 16, 17, 18, 19, or 20 contiguous nucleotides of the targeting sequence of a guide RNA provided in the Table 1A. [0157] For example, Spy Cas9 guides can be 16-, 17-, preferably 18-, 19-, or 20- nucleotides in length, such that, in some embodiments, the Spy Cas9 targeting sequence comprises 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250- 269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686- 705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845- 1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073- 2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891- 2910, 2956-2975, or 2982-3001, optionally from nucleotides 153-173, 153-172, 154-173, or 250- 269 of SEQ ID NO: 1, or a reverse complement thereof; for example, 16, 17, 18, 19, or 20 contiguous nucleotides of the targeting sequence of a guide RNA provided in the Table 1A, e.g., a targeting site of a guide RNA selected from S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6. Table 1A: HBV Spy-Cas9 guide sequences and sequence coordinates
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
[0158] As shown in Table 1B, the guide targeting sequences for gRNA to be used with a Spy-Cas9 show significant cross-reactity to different strains of HBV. As such, a single gRNA may be suitable for Spy-Cas9-mediated editing of the genomes of more than one HBV strains. However, in the event of an infection with an unknown strain of HBV, multiple gRNAs can be administered to increase the likelihood that the unknown HBV strain genome will be edited.
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
[0159] In some embodiments, the targeting sequence is in an open reading frame or in a genome, e.g., a viral genome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%, or is 100%. For example, in some embodiments, the targeting sequence comprises a sequence that is at least 80%, 85%, preferably 90%, or 95%, or is 100% identical or complementary to 20 contiguous nucleotides of a sequence selected from nucleotides 163- 466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 57-76, 66- 85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072- 2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001, optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; for example, at least 80%, 85%, preferably 90%, or 95%, or is 100% identical to a targeting sequence of a guide RNA provided in the Table 1A, e.g., a targeting site of a guide RNA selected from S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6. In some embodiments, the targeting sequence and the target region may be 100% complementary or identical to at least 20 contiguous nucleotides of nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364- 383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697- 716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880- 1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982- 3001; optionally from nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1, or to a targeting sequence of a guide RNA provided in the Table 1A, e.g., a target site of a guide RNA with an HBV number of S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, or S93, optionally S1, S5, or S6. In other embodiments, the guide sequence and the target region may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches within the duplex formed between the guide and the target sequence, where the total length of the target sequence is 16, 17, 18, 19, 20 nucleotides, or more. In some embodiments, the guide sequence and the target sequence may contain 1-4 mismatches where the guide sequence comprises at least 20 nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. That is, the guide sequence and the target region may form a duplex region having 16, 17, 18, 19, 20 base pairs, or more. In certain embodiments, the duplex region may include 1, 2, 3, or 4 mismatches such that guide strand and target sequence are not fully complementary. For example, a guide strand and target sequence may be complementary over a 20 nucleotide region, including 2 mismatches, such that the guide sequence and target sequence are 90% complementary providing a duplex region of 18 base pairs out of 20. More tolerated mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Sternberg et al., 2015, Nature:527:110-113) [0160] For example, Nme guide targeting sequences can be 19, 20, 21, preferably 22, 23, or 24 nucleotides in length such that, in some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447- 470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1, or a reverse complement thereof; for example, 19, 20, 21, preferably 22, 23, or 24 contiguous nucleotides of a targeting sequence of a guide RNA provided in the Table 2A, e.g., a target site from a guide with an HBV number of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37. Table 2A: HBV Nme-Cas9 guide sequences and sequence coordinates
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
[0161] As shown in Table 2B, the guide targeting sequences for gRNA to be used with an Nme-Cas9 show significant crossreactity to different strains of HBV. As such, a single gRNA may be suitable for Nme-Cas9-mediated editing of the genomes of more than one HBV strains. However, in the event of an infection with an unknown strain of HBV, multiple gRNAs can be administered to increase the likelihood that the unknown HBV strain genome will be edited.
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
[0162] In some embodiments, the target sequence is in an open reading frame or a in a genome, e.g., a viral genome, and is complementary to the targeting sequence. In some embodiments, the degree of complementarity or identity between a targeting sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%. For example, in some embodiments, the guide sequence comprises a sequence 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, 1821-2460, for example, nucleotides 35-58, 146-169, 153-176, 216-239, 330- 356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1, or a reverse complement thereof; or of a targeting sequence of a guide RNA provided in the Table 2A, e.g., a target site from a guide with an HBV number of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37. In some embodiments, the targeting sequence and the target sequence may be 100% complementary or identical. In other embodiments, the targeting sequence and the target sequence may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the targeting sequence and the target sequence may contain 1-2, preferably no more than 1 mismatch, where the total length of the target sequence is 19, 20, 21, 22, preferably 23, or 24, nucleotides, or more. In some embodiments, the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises at least 24 nucleotides. In some embodiments, the targeting sequence and the target sequence may contain 1-2 mismatches where the targeting sequence comprises 24 nucleotides. That is, the targeting sequence and the target sequence may form a duplex region having 24 base pairs, or more. In certain embodiments, the duplex region may include 1-2 mismatches such that targeting sequenceand target sequence are not fully complementary. Mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Edraki et al., 2019. Mol. Cell, 73:1-13). [0163] Target sequences for RNA-guided DNA binding agents, as defined by the targeting sequence of a guide RNA, may be present on either the positive or negative strand. Tables and other disclosures provided herein may recite genomic coordinates or position within a nucleotide sequence as a target sequence. It is understood that the guide can be complementary to either the positive or negative strand of the DNA as defined by the genomic coordinates or position within a nucleotide sequence. The sequence to which the guide is complementary depends on the presence of an appropriate PAM for the RNA guided DNA binding protein on the opposite strand. Thus, in some embodiments, when the targeting sequence binds the reverse complement of a target sequence, i.e., the targeting sequence is identical to certain nucleotides of the sense (positive) strand of the target sequence, when the PAM is present in the sense strand, except for the substitution of U for T in the targeting sequence. [0164] As used herein, an “RNA-guided DNA binding agent” means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the presence of a PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease,” as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas nickases include nucleases in which one of the RuvC or HNH domain of the Cas protein, such that only a single strand is cleaved by the nuclease The dCas DNA binding agent may be a dead nuclease comprising non-functional nuclease domains (RuvC or HNH domain). In some embodiments the Cas cleavase or Cas nickase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g., via fusion with a FokI domain. Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. [0165] As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases/nickases (e.g., H840A, or D10A variants of Spy Cas9 and D16A and H588A of Nme Cas9, e.g., Nme2 Cas9), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). [0166] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided below. Methods for identifying alternate nucleotide sequences encoding Cas9 polypeptide sequences, including alternate naturally occurring variants, are known in the art. Sequences with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 nucleic acid sequences, or nucleic acid sequences encoding the amino acid sequences provided herein are also contemplated. In certain embodiments, the nucleotide sequence encoding the Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Sequences with at least 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 amino acid sequences provided herein are also contemplated. In certain embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence. [0167] Exemplary open reading frames and amino acid sequences for Cas9 (SEQ ID NO: 705, 706, 707, 710, 711, 712, 713, 715, 716, 718, 719, 721, 722, 729, 730) and uracil glycosylase inhibitors (SEQ ID NO: 708, 709, 724, 725, 732, 733) are provided in Table 40. [0168] As used herein, a “cytidine deaminase” means a polypeptide or complex of polypeptides that is capable of cytidine deaminase activity, that is catalyzing the hydrolytic deamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine. Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol.22:367-77, 2005; Conticello, Genome Biol.9:229, 2008; Muramatsu et al., J. Biol. Chem.274: 18470-6, 1999); Carrington et al., Cells 9:1690 (2020)). In some embodiments, variants of any known cytidine deaminase or APOBEC protein are encompassed. Variants include proteins having a sequence that differs from wild-type protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For instance, a shortened sequence could be used, e.g., by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to a reference sequence. The variant is “functional” in that it shows a catalytic activity of DNA editing. [0169] As used herein, the term “APOBEC3A” refers to a cytidine deaminase such as the protein expressed by the human A3A gene. The APOBEC3A may have catalytic DNA editing activity. An amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 734. In some embodiments, the APOBEC3A protein is a human APOBEC3A protein or a wild-type protein. Variants include proteins having a sequence that differs from wild-type APOBEC3A protein by one or several mutations (i.e., substitutions, deletions, insertions), such as one or several single point substitutions. For instance, a shortened APOBEC3A sequence could be used, e.g. by deleting N-terminal, C-terminal, or internal amino acids, preferably one to four amino acids at the C- terminus of the sequence. As used herein, the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to an APOBEC3A reference sequence. The variant is “functional” in that it shows a catalytic activity of DNA editing. In some embodiments, an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence). [0170] Several Cas9 orthologs have been obtained from N. meningitidis (Esvelt et al., Nat. Meth., vol.10, 2013, 1116 - 1121; Hou et al., PNAS, vol.110, 2013, pages 15644 - 15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 ortholog functions efficiently in mammalian cells, recognizes an N4CC PAM, and can be used for in vivo editing (Ran et al., Nature, vol.520, 2015, pages 186 - 191; Kim et al., Nat. Commun., vol.8, 2017, pages 14500). Nme2Cas9 has been shown to be naturally resistant to off-target editing (Lee et al., Mol. Ther., vol.24, 2016, pages 645 - 654; Kim et al., 2017). See also e.g., WO/2020081568 (e.g., pages 28 and 42), describing an Nme2Cas9 D16A nickase, the contents of which are hereby incorporated by reference in its entirety. Throughout, “NmeCas9” or “Nme Cas9” is generic and an encompasses any type of NmeCas9, including, Nme1Cas9, Nme2Cas9, and Nme3Cas9. [0171] As used herein, the term “fusion protein” refers to a hybrid polypeptide which comprises polypeptides from at least two different proteins or sources. One polypeptide may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy- terminal (C- terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. [0172] As used herein, the term “uracil glycosylase inhibitor,” “uracil-DNA glycosylase inhibitor” or “UGI” refers to a protein that is capable of inhibiting a uracil-DNA glycosylase (UDG) base-excision repair enzyme (e.g., UniPROT ID: P14739; SEQ ID NO: 735). [0173] The term “linker,” as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). Exemplary peptide linkers are disclosed elsewhere herein. Exemplarly linkers for use in gRNAs are disclosed elsewhere herein. [0174] “Modified uridine” is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton. In some embodiments, a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine, e.g., N1-methyl-psuedouridine. [0175] “Uridine position” as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide in which “100% of the uridine positions are modified uridines” contains a modified uridine at every position that would be a uridine in a conventional RNA (where all bases are standard A, U, C, or G bases) of the same sequence. Unless otherwise indicated, a U in a polynucleotide sequence of a sequence table or sequence listing in or accompanying this disclosure can be a uridine or a modified uridine. [0176] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with the target sequence and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking. [0177] As used herein, an “RNA-guided DNA binding agent” means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavases (which have double strand cleaving activity), Cas nickases (which have single strand cleaving activity), and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease,” as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. The dCas DNA binding agent may be a dead nuclease comprising non-functional nuclease domains (RuvC or HNH domain). In some embodiments the Cas cleavase or Cas nickase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g., via fusion with a FokI domain. In some embodiments, the RNA-guided DNA binding agent has nuclease activity, e.g., cleavase or nickase activity. [0178] As used herein, a “control” is understood as an appropriate matched sample or subject for comparison. For example, a control can be a cell population treated in the same manner as the test population except that the treatment used for the control population lacks at least one active agent, e.g., a guide RNA, an mRNA encoding a nuclease, an insertion construct, a lipid formulation. In certain embodiments, a control may be an internal control, e.g., a cell population or subject prior to treatment. [0179] In certain embodiments, a “control” as in a control subject is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease. In certain embodiments, a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention. In certain embodiments, a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treated subject, to provide a normal control, e.g., an enzyme concentration or activity in a subject sample. In certain embodiments, a normal control can be a population control, i.e., the average of subjects in the general population. In certain embodiments, a control can be an untreated subject with the same disease. In certain embodiments, a control can be a subject treated with a different therapy, e.g., the standard of care. In certain embodiments, a control can be a subject or a population of subjects from a natural history study of subjects with the disease of the subject being compared. In certain embodiments, the control is matched for certain factors to the subject being tested, e.g., age, gender. In certain embodiments, a control may be a control level for a particular lab, e.g., a clinical lab. The ability to design or select appropriate controls is within the ability of those of skill in the art. It is understood when relative values are provided, they can be considered as relative values as compared to an appropriate control. [0180] As used herein, “purified” such as in “purified protein” or “purified nucleic acid” is understood as being substantially removed from the mixture in which it was made, e.g., a cell, a subject sample, a reaction mixture. In certain embodiments, purified is understood as at least 50% of the mixture is the purified compound, e.g., purified protein or purified nucleic acid by weight. In certain embodiments, purified is understood as at least 80% of the mixture is the purified compound by weight. [0181] As used herein, “subject” includes primates, including human and non-human primates, mouse, and rat. In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a non-human subject. In certain embodiments, the subject is a non-human subject expressing one or more human genes, e.g., a transgenic mouse expressing a human gene, a mouse in which the liver has been repopulated with human hepatocytes (e.g., PXB-mouse®). Such models are well known in the art. [0182] As used herein, a “target sequence” refers to a sequence of nucleic acid in a target genome or open reading frame, in either the positive or the negative strand, that has complementarity to the guide sequence of the gRNA, i.e., that is sufficiently complementary to the guide sequence to permit specific binding of the guide sequence. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence. The specific length of the target sequence and the number of mismatches possible between the target sequence and the guide sequence depend, for example, on the identity of the Cas nuclease being directed by the gRNA. [0183] As used herein, a first sequence is considered to be “identical” or have “100% identity” with a second sequence if an alignment of the first sequence to the second sequence shows that all of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAG has 100% identity to the sequence AAGA because an alignment would give 100% identity in that there are matches, without gaps, to all three positions of the first sequence. Less than 100% identity can be calculated using routine methods. For example ACG would have 67% identity with AAGA as two of the three positions of the first sequence are matches to the second sequence (2/3 = 67%). The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith–Waterman and Needleman–Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate. [0184] Similarly, as used herein, a first sequence is considered to be “fully complementary” or 100% complementary” to a second sequence when all of the nucleotides of a first sequence are complementary to a second sequence, without gaps. For example, the sequence UCU would be considered to be fully complementary to the sequence AAGA as each of the nucleobases from the first sequence base pair with the nucleotides of the second sequence, without gaps. The sequence UGU would be considered to be 67% complementary to the sequence AAGA as two of the three nucleobases of the first sequence base pair with nucleobases of the second sequence. One skilled in the art will understand that algorithms are available with various parameter settings to determine percent complementarity for any pair of sequences using, e.g., the NCBI BLAST interface (blast.ncbi.nlm.nih.gov/Blast.cgi) or the Needleman-Wunsch algorithm. [0185] “Messenger RNA” or “mRNA” is used herein to refer to a polynucleotide that comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise one or more chemically modified nucleosides such as 5-methyl-cytidine (5mC), 2-thio-uridine (2sU),N1-methylpseudouridine (m1 ΨU) and pseudo-uridine ( ΨU), or a modified cap structure as provided below. [0186] Exemplary guide sequences useful in the guide RNA compositions and methods described herein are shown in Tables 1A and 2A and throughout the application. For example, where Tables 1A and 2A show a guide sequence, this guide sequence may be used in a guide RNA to direct a RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9, to a target sequence. Target sequences, as provided in Tables 1A and 2A by their positions within SEQ ID NO: 1, include both the positive and negative strands corresponding to the indicated DNA sequence in SEQ ID NO: 1 (i.e., the sequence given and the sequence’s reverse complement). In some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence, except for the substitution of U for T in the guide sequence. [0187] As used herein, “indels” refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in a target nucleic acid. As used herein, when indel formation results in an insertion, the insertion is a random insertion at the site of a double stranded break and is not directed by or based on a template sequence. [0188] As used herein, “inhibit expression” and the like refer to a decrease in expression (e.g., knockdown or knockout) of a particular gene product (e.g., protein, mRNA, or both). Expression of a protein (i.e., gene product) can be measured by detecting total cellular amount of the protein from a tissue sample, e.g., biopsy, or cell population of interest by detecting expression of a protein as individual members of a population of cells, e.g., by cell sorting to define percent of cells expressing a protein, or expression of a protein in cells in aggregate, e.g., by ELISA or western blot, including ELISA or western blot performed on a body fluid, e.g, blood or serum or plasma derived therefrom, into which the expressed protein is secreted. Inhibition of expression can result from genetic modification of a gene sequence, e.g., a genomic sequence, such that the full-length gene product, or any gene product, is no longer detected, e.g. knockdown of the gene. Certain genetic modifications can result in the introduction of frameshift or nonsense mutations that prevent translation of the full-length gene product. Genetic modifications at a splice site, e.g., at a position sufficiently close to a splice acceptor site or a splice donor site to disrupt splicing, can prevent translation of the full-length protein. Inhibition of expression can result from a genetic modification in a regulatory sequence that is required for the expression of the gene product (e.g., a promoter sequence, a 3’ UTR sequence, a capping sequence, a 5’ UTR sequence, a poly A sequence, and the like). Inhibition of expression may also result from disrupting expression or activity of regulatory factors required for translation of the gene product, e.g., production of no gene product. For example, a genetic modification in a transcription factor sequence, inhibiting expression of the full-length transcription factor, can have downstream effects and inhibit expression of one or more gene products controlled by the transcription factor. Therefore, inhibition of expression can be predicted by changes in genomic or mRNA sequences. As used herein, inhibit expression can be understood to inhibit expression of protein activity, even if the protein is expressed. For example a mutation in a polymerase can inhibit activity of the polymerase without reducing the level of expression of the polymerase, e.g., as determined by a protein detection assay, e.g., ELISA or western blot. For example, as used herein, inhibition of expression of an HBV polymerase can be determined by reduction of HBV polymerase activity, e.g., using methods provided herein, and does not necessarily require a lower level of protein as determined by a protein detection assay. As used herein, inhibition of protein activity does not require complete inhibition of protein activity. For example, inhibition of HBV polymerase activity can result in a decreased in HBV DNA, e.g., a therapeutically relevant decrease in HBV DNA. [0189] Mutations expected to result in inhibition of expression can be detected by known methods including next generation sequencing of DNA isolated from a tissue sample or cell population of interest. Inhibition of expression can be determined as the percent of cells in a population having a predetermined level of expression of a protein, i.e., a reduction of the percent or number of cells in a population expressing a protein of interest at least a certain level. Inhibition of expression can also be assessed by determining a decrease in overall protein level, e.g., in a cell or tissue sample, e.g., a biopsy sample. Mutations that disrupt protein function without a corresponding decrease in protein level, e.g., disrupt polymerase activity, may or may not be predictable from known sequences or structures and may need to be identified empirically. In certain embodiments, inhibition of expression of a secreted protein can be assessed in a fluid sample, e.g., cell culture media or a body fluid. Proteins may be present in a body fluid, e.g., blood or urine, to permit analysis of protein level. In certain embodiments, protein level may be determined by protein activity or the level of a metabolic product, e.g., in urine or blood. In some embodiments, “inhibition of expression” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of an mRNA or a protein expressed in a tissue sample or by a population of cells. In some embodiments, “inhibition” may refer to some loss of expression of a particular gene product, for example at the cell surface or secreted into a bodily fluid, e.g., blood. In some embodiments, “inhibition” may refer to some loss of expression in one, or more, cell or tissue types, but not all cell or tissue types, e.g., inhibition of expression in liver, but not in other organs. It is understood that the level of inhibition of expression is relative to a starting level in the same type of subject sample. For example, routine monitoring of a protein level is more easily performed in a fluid sample from a subject, e.g., blood or urine, than in a tissue sample, e.g., a biopsy sample. In certain embodiments, a correlation is known, or established, wherein the level of a biomarker, e.g., in blood or urine, is correlated with the level of inhibition of expression of a target gene. It is understood that the level of inhibition of expression is for the sample being assayed. Similarly, in animal studies where serial tissue samples may be obtained, e.g., liver tissue, the target may be expressed in other tissues. Therefore, the level of inhibition of expression is not necessarily the level of inhibition of expression systemically, but within the tissue, cell type, or fluid being sampled. [0190] As used herein, a “genetic modification” is a change at the DNA level, e.g. induced by a CRISPR/Cas gRNA and Cas system, e.g., a CRISPR/Cas9 gRNA and Cas9 system. A genetic modification may comprise an insertion, deletion, or substitution (i.e., base sequence substitution, i.e., mutation), typically within a defined sequence or genomic locus. A genetic modification changes the nucleic acid sequence of the DNA. A genetic modification may be at a single nucleotide position. A genetic modification may be at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides. In the context of the overlapping HBV genome, the genetic modification may be at multiple overlapping ORF in the genome. A genetic modification can be in a coding sequence, e.g., an open reading frame (ORF). As used herein, a genetic modification can be used to prevent translation of an endogenous full-length protein having an amino acid sequence of the full-length protein prior to genetic modification of the defined sequence or genomic locus. Prevention of translation of a full-length protein or gene product includes prevention of translation of a protein or gene product of any length. Translation of an endogenous full-length protein can be prevented, for example, by a frameshift mutation that results in the generation of a premature stop codon or by generation of a nonsense mutation. A genetic modification can include a modification that inhibits protein activity that may not correspondingly decrease protein level as determined by a protein detection assay, e.g., ELISA or western blot. [0191] “Treatment” as used herein is understood as reducing at least one sign or symptom of the disease or indication. Reduction can include to a frequency or severity such that the sign or symptom of the disease is no longer detectable. Treatment can include administration of more than one dose of the agent. For example, in some embodiments, treatment can include administration of 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. Treatment can include administration with other agents. Effective treatment does not require a cure or complete elimination of the disease or indication. The rate of progression or development of a disease can be compared to the progression or development of a disease in an appropriately matched control, e.g., a population control, a control from a natural history study. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation of one or more signs or symptoms in a subject with HBV infection including, but not limited to, the presence of serum HBV DNA or liver HBV ccc DNA, the presence of serum or liver HBV antigen, e.g., HBsAg or HBeAg. Presence or levels of signs or symptoms can also be used to monitor therapeutic effects of treatment or determine a course of treatment (e.g., to determine the administration or amount of one or more doses or further doses). For example, in some embodiments, after administering a first dose of a guide RNA or composition provided herein to a subject with HBV infection, a clinical marker, e.g., serum HBsAg level, is mearured, and a subsequent dose is administered if the serum HBsAg is greater than a threshold value, optionally wherein the threshold value is 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml. Diagnostic criteria for HBV infection are well known in the art. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment, or lower risk of HCC development. [0192] In certain embodiments, treatment of HBV infection results in a “functional cure” of hepatitis B. As used herein, the term “functional cure” is understood to be clearance of circulating HBsAg and is preferably accompanied by conversion to a status in which HBsAg antibodies become undetectable using a clinically relevant assay. For example, undetectable antibodies can include a signal lower than 10 mIU/ml as measured by Chemiluminescent Microparticle Immunoassay (CMIA) or any other immunoassay, and may be called anti-HBs seroconversion. Functional cure does not require clearance of all replicative forms of HBV (e.g., cccDNA from the liver). Anti-HBs seroconversion occurs spontaneously in about 0.2-1% of chronically infected patients per year. However, even after anti-HBs seroconversion, low level persistence of HBV is observed for decades indicating that a functional rather than a complete cure occurs. Without being bound to mechanism, it is proposed that the immune system is able to keep HBV in check. A functional cure permits discontinuation of any treatment for the HBV infection. However, it is understood that a “functional cure” for HBV infection may not be sufficient to prevent or treat diseases or conditions that result from HBV infection, e.g., liver fibrosis, HCC, cirrhosis. [0193] In certain embodiments, treatment of HBV infection results in a “sterilizing cure” of hepatitis B. As use herein, the term “sterilizing cure” is understood as clearance of all circulating HBsAg, is preferably accompanied by conversion to a status in which HBsAg antibodies become undetectable using a clinically relevant assay, and clearance of all detectable replicative forms of HBV (e.g., cccDNA from the liver) as determined by a clinically relevant assay (e.g., analysis of a liver biopsy). [0194] As used herein, “delivering” and “administering” are used interchangeably. [0195] “Co-administration”, as used herein, means that a plurality of substances are administered sufficiently close together in time so that the agents act together. Co- administration encompasses administering substances together in a single formulation and administering substances in separate formulations close enough in time so that the agents act together. [0196] As used herein, the phrase “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable and that are not otherwise unacceptable for pharmaceutical use. Pharmaceutically acceptable generally refers to substances that are non-pyrogenic. Pharmaceutically acceptable can refer to substances that are sterile, especially for pharmaceutical substances that are for injection or infusion. II. Brief Description of the Drawings [0197] Figure 1 is a graph showing percent editing of a hepatitis B coding sequence present in the HepAD38 cell line as determined by next generation sequencing (NGS). Editing (C-to-T%, C-to-A/G%, and indel formation) and predicted editing outcomes (STOP Gained or START Lost) for Spy-Cas9 guides targeting HBV S-antigen tested with a fixed dose of sgRNA in combination with mRNA(s) encoding either Spy-BC22n and UGI or Spy- cleavaseare shown. [0198] Figure 2 is a graph showing percent editing of a hepatitis B coding sequence present in the HepAD38 cell line as determined by NGS. Editing and predicted editing outcomes for Nme2-Cas9 guides targeting HBV S-antigen tested with a fixed dose of sgRNA in combination with mRNA(s) encoding Nme2-BC22n and UGI or Nme2-cleavase are shown. [0199] Figure 3A and 3B are graphs showing percent C-to-T editing of a hepatitis B coding sequence present in theHepAD38 cell line as determined by NGS. Cells were contacted with guides targeting HBV S-Antigen at 2-fold serial dilutions from 100 nM to 1.56 nM, with a fixed concentration of mRNAs encoding (A) Spy-BC22n and UGI, or (B) Nme2-BC22n and UGI. Figure 3A shows percent C-to-T editing against doses of Spy- BC22n guide RNAs. Figure 3B shows percent C-to-T editing against doses of Nme2-BC22n guide RNAs. [0200] Figures 4A and 4B are graphs showing percent C-to-T editing of a hepatitis B coding sequence present in theHepAD38 cell line as determined by NGS. Cells were contacted with guides targeting HBV S-Antigen at 2-fold serial dilutions from 10 nM to 0.156 nM, with a fixed concentration of mRNAs encoding (A) Spy-BC22n and UGI, or (B) Nme2-BC22n and UGI. Figure 4A shows percent C-to-T editing against doses of Spy- BC22n guide RNAs. Figure 4B shows percent C-to-T editing against doses of Nme2-BC22n guide RNAs. [0201] Figure 5A and 5B show the percent knockdown of HBsAg levels relative to control guide RNA for Nme2 base editor guide RNAs. Figure 5A shows the total intracellular HBsAg and the total amount of secreted HBsAg. Figure 5B shows the percent intracellular HBV Copy Number (CN) knockdown for primer set 1 and primer set 2. Dotted line indicates the level of 90% knockdown. [0202] Figure 6A and 6B show the percent knockdown of HBsAg levels relative to control guide RNA for Spy base editor guide RNA. Figure 6A shows and total intracellular HBsAg and the total amount of secreted HBsAg. Figure 6B shows the percent intracellular HBV Copy Number (CN) knockdown for primer set 1 and primer set 2. [0203] Figure 7 shows the relative percent HBsAg knockdown relative to LNP-GFP mRNA in PHH. Guides were screened at 11, 33, or 100 ng/well. [0204] Figure 8 shows the time course for the Log10 change of HBsAg level from the baseline over a period of 5 weeks post LNP injection using either Spy-BC22n or Nme2- BC22n guides. [0205] Figure 9 shows the Log10 change in serum knockdown of total HBV DNA at five weeks post LNP injection with either Spy-BC22n or Nme2-BC22n guides. [0206] Figure 10 shows the correlation between HBsAg and total HBV DNA levels in serum at five weeks post LNP injection relative to AAV only. Correlation is shown to be very high with an R2 value of 0.9652. [0207] Figure 11 shows the C-to-T editing as determined by NGS. Cells were treated with either Nme2-BC22n or Spy-BC22n guides targeting HBV in a dose response curve using a 3-fold dilution scheme with concentrations ranging from 50 nM to 0 nM. [0208] Figure 12 shows the percent of secreted HBsAg knockdown using the two highest doses from the dose response curve of either a 50 nM or 16 nM dose of either Spy- BC22n or Nme2-BC22n guides. [0209] Figure 13 shows the percent knockdown of secreted HBsAg in HepAD38 cells targeted by multiplex combinations of Nme2-BC22n HBV guides measured by MSD assay. [0210] Figure 14 shows the percent knockdown of secreted HBsAg in HepAD38 cells targeted by additional multiplex combinations of Nme2-BC22n HBV guides measured by MSD assay. [0211] Figure 15 shows the percent knockdown of secreted HBsAg with single or multiplexed HBV guides. [0212] Figure 16 shows the percent total HBV DNA copy number knockdown with single or multiplexed HBV guides. [0213] Figure 17 shows the HBsAg concentration (ng/ml) following treatment with guides with different modification patterns. [0214] Figure 18 shows the mean HBsAg knockdown IC50 (nM) with guides with different modification patterns. [0215] Figure 19 shows the serum HBsAg log10 reduction time course relative to individual animal baseline. [0216] Figure 20 shows the log10 knockdown of total serum HBV DNA relative to TSS group at week 6. [0217] Figure 21A shows the serum HBsAg log10 reduction time course relative to individual animal baseline with HBV genotype A. [0218] Figure 21B shows the serum HBsAg log10 reduction time course relative to individual animal baseline with HBV genotype C. [0219] Figure 22 shows the endpoint comparisons of log10 serum HBsAg reduction from baseline. [0220] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. III. Compositions Compositions Comprising Guide RNA (gRNAs) [0221] Provided herein are compositions useful for altering a DNA sequence, e.g., inducing a single-stranded (SSB) or double-stranded break (DSB), within an HBV ORF, e.g., within nucleotides 163-466 of SEQ ID NO: 1, or also within nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, e.g., using a guide RNA with an RNA-guided DNA binding agent (e.g., a CRISPR/Cas system). Guide sequences targeting an HBV ORF are shown in Tables 1A and 2A, as are the target sites within SEQ ID NO: 1 that such guide RNA targets. [0222] A nucleic acid sequence of a representative HBV genome is provided as SEQ ID NO: 1: SEQ ID NO: 1 CAGTGGAATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTT CCCTGCTGGTGGCTCCAGTTCAGGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTAT CGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAACATGGAGAACATCACATCAGGATTC CTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACC GCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTG GCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCT GGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCT CATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAG GATCCTCAACCACCAGCACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGAACCTCT ATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCC ATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCA GTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTT ATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCT GTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAACAAAGAGATGGGG TTACTCTCTGAATTTTATGGGTTATGTCATTGGAAGTTATGGGTCCTTGCCACAAGAACACA TCATACAAAAAATCAAAGAATGTTTTAGAAAACTTCCTATTAACAGGCCTATTGATTGGAAA GTATGTCAACGAATTGTGGGTCTTTTGGGTTTTGCTGCCCCATTTACACAATGTGGTTATCC TGCGTTAATGCCCTTGTATGCATGTATTCAATCTAAGCAGGCTTTCACTTTCTCGCCAACTT ACAAGGCCTTTCTGTGTAAACAATACCTGAACCTTTACCCCGTTGCCCGGCAACGGCCAGGT CTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGTCATGGGCCATCAGCG CGTGCGTGGAACCTTTTCGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTT TTGCTCGCAGCAGGTCTGGAGCAAACATTATCGGGACTGATAACTCTGTTGTCCTCTCCCGC AAATATACATCGTATCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTC CTTTGTTTACGTCCCGTCGGCGCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGAC TCTCTCGTCCCCTTCTCCGTCTGCCGTTCCGACCGACCACGGGGCGCACCTCTCTTTACGCG GACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGT CGCATGGAGACCACCGTGAACGCCCACCGAATGTTGCCCAAGGTCTTACATAAGAGGACTCT TGGACTCTCTGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGACTGTTTGTTTAAAG ACTGGGAGGAGTTGGGGGAGGAGATTAGATTAAAGGTCTTTGTACTAGGAGGCTGTAGGCAT AAATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCA TGTCCTACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACCC TTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCTTCTGACTTCTTTCCTT CAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAG CATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGAC TCTAGCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTT ATGTCAACACTAATATGGGCCTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTC ACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTGTCTTTCGGAGTGTGGATTCGCACTCC TCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACTACTGTTGTTA GACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCG CCGCGTCGCAGAAGATCTCAATCTCGGGAACCTCAATGTTAGTATTCCTTGGACTCATAAGG TGGGGAACTTTACTGGTCTTTATTCTTCTACTGTACCTGTCTTTAATCCTCATTGGAAAACA CCATCTTTTCCTAATATACATTTACACCAAGACATTATCAAAAAATGTGAACAGTTTGTAGG CCCACTTACAGTTAATGAGAAAAGAAGATTGCAATTGATTATGCCTGCTAGGTTTTATCCAA AGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACCTTATTATCCAGAACATCTAGTT AATCATTACTTCCAAACTAGACACTATTTACACACTCTATGGAAGGCGGGTATATTATATAA GAGAGAAACAACACATAGCGCCTCATTTTGTGGGTCACCATATTCTTGGGAACAAGATCTAC AGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCACCAGTTGGA TCCAGCCTTCAGAGCAAACACAGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACACCT GGCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGCTGGGTTTCACCCCACCGCACGGA GGCCTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCC TCCTGCCTCCACCAATCGCCAGACAGGAAGGCAGCCTACCCCGCTGTCTCCACCTTTGAGAA ACACTCATCCTCAGGCCATGCAGTGG [0223] Guide sequences targeted to sites adjacent to an appropriate PAM, e.g., a Spy Cas9 PAM, e.g., as shown in Table 1A may further comprise additional nucleotides, which can be referred to as a scaffold sequence or a conserved portion, to form a crRNA or a crRNA joined to a trRNA to form a sgRNA e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3’ end (Table 3A). The #mer refers to the length of the crRNA or the sgRNA when a 20 nucleotide guide targeting sequence is included 5’ to the scaffold sequence provided in Table 3A. Table 3A: Exemplary Unmodified Spy Scaffold Sequences
Figure imgf000064_0001
Figure imgf000065_0001
Table 3B: Exemplary Unmodified Spy Guide RNA Sequences
Figure imgf000065_0002
Wherein the Ns collectively are a guide sequence provided herein. [0224] In the case of a sgRNA, the guide sequences may be integrated into the following modified guide scaffold motifs (Table 4). The #mer refers to the length of the sgRNA when a 20 nucleotide guide targeting sequence, either a modified or unmodified sequence, is included 5’ to the scaffold sequence provided in Table 4: Table 4: Exemplary Modified Spy Guide Scaffold Sequences^
Figure imgf000066_0001
wherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and phosphodiesterase linkage. [0225] In certain embodiments, the guide sequence is a chemically modified sequence. In certain embodiments, the chemically modified guide sequence is (mN*)3(N)13- 17. In certain embodiments, the guide sequence is (mN*)3(N)17, i.e., mN*mN*mN*NNNNNNNNNNNNNNNNN. In certain embodiments, each N of the (N)13- 17 or the (N)17 is unmodified. In certain embodiments, the each N in the (N)13-17 or the (N)17 is independently modified, e.g., independently modified with a 2’-O-methyl modification. ^^^^^^ In the case of a sgRNA, the guide sequences may further comprise a SpyCas9 sgRNA scaffold sequence. An example of a SpyCas9 sgRNA scaffold sequence is shown in the Table 11 below (SEQ ID NO: 268: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGC – “Exemplary SpyCas9 sgRNA-1”), included at the 3’ end of the guide sequence, and provided with the domains as shown in the table below. LS is lower stem. B is bulge. US is upper stem. H1 and H2 are hairpin 1 and hairpin 2, respectively. Collectively H1 and H2 are referred to as the hairpin region. A model of the structure containing both a guide sequence and a scaffold sequence is provided in Figure 10A of WO2019237069, which is incorporated herein by reference. ^ [0227] The nucleotide sequence of Exemplary SpyCas9 sgRNA-1 may serve as a template sequence for specific chemical modifications, sequence substitutions and truncations. [0228] In certain embodiments, the gRNA is an sgRNA or a dgRNA, for example, and it optionally comprises a chemical modification. In some embodiments, the modified sgRNA comprises a guide sequence and a SpyCas9 sgRNA sequence, e.g., Exemplary SpyCas9 sgRNA-1. A gRNA, such as an sgRNA, may include modifications on the 5’ end of the guide sequence or on the 3’ end of the SpyCas9 sgRNA sequence, such as, e.g., Exemplary SpyCas9 sgRNA-1 at one or more of the terminal nucleotides, e.g., at 1, 2, 3, or 4 of the nucleotides at the 3’ end or at the 5’ end. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2- methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or an inverted abasic modified nucleotide; or a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. In certain embodiments, the modified nucleotide includes a PS linkage. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage. [0229] In certain embodiments, using SEQ ID NO: 268: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGC “Exemplary SpyCas9 sgRNA-1,” see WO2019237069, the contents of which are incorporated herein by reference). The portions of the Exemplary SpyCas9 sgRNA-1 and position numbering scheme are set forth in Table 11 below. [0230] As an example, the Exemplary SpyCas9 sgRNA-1 further includes one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 268); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region, wherein 1. the modified nucleotide is optionally selected from a 2’-O-methyl (2’- OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or a combination thereof; or 2. the modified nucleotide optionally includes a 2’-OMe modified nucleotide. [0231] Guide sequences targeted to sites adjacent to an appropriate PAM, e.g., an NmeCas9 PAM, e.g., as shown in Table 2A may further comprise additional nucleotides to form a crRNA or a crRNA joined to a trRNA to form a sgRNA e.g., with the exemplary nucleotide sequence following the guide sequence at its 3’ end as provided in Table 5. The portions of the Exemplary NmeCas9 sgRNA and position numbering scheme, including both a guide sequence and a scaffold sequence, are set forth in Table 13 below. [0232] In certain embodiments, using SEQ ID NO: 455 (“Exemplary NmeCas9 sgRNA-1”), as an example, the Exemplary NmeCas9 sgRNA-1 includes: A. A guide RNA (gRNA) comprising a guide region and a conserved region, the conserved region comprising one or more of: (a) a shortened repeat/anti-repeat region, wherein the shortened repeat/anti- repeat region lacks 2-24 nucleotides, wherein (i) one or more of nucleotides 37-48 and 53-64 is deleted and optionally one or more of nucleotides 37-64 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2-10, optionally 2-8 nucleotides, wherein (i) one or more of nucleotides 82-86 and 91-95 is deleted and optionally one or more of positions 82-96 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18, optionally 2-16 nucleotides, wherein (i) one or more of nucleotides 113-121 and 126-134 is deleted and optionally one or more of nucleotides 113-134 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; wherein one or both nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 455; wherein, optionally, at least 10 nucleotides are modified nucleotides. [0233] Exemplary unmodified conserved nucleotide sequences, also referred to as scaffold sequences, are shown in Table 5. The #mer refers to the length of the sgRNA when a 24 nucleotide guide targeting sequence is included 5’ to the scaffold sequence provided in Table 5. Table 5: Unmodified Nme Conserved Region Nucleotide Sequences
Figure imgf000070_0001
[0234] In the case of a sgRNA, modified guide sequences may be integrated into one of the following exemplary modified conserved portion motifs (Table 6). The #mer refers to the length of the sgRNA when a 24 nucleotide guide targeting sequence, either a modified or unmodified sequence, is included 5’ to the scaffold sequence provided in Table 6: Table 6: Exemplary Modified Conserved Region Nme Guide RNA Motifs
Figure imgf000071_0001
[0235] wherein “m” indicates a 2’-O-Me modification, and a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and a phosphorothioate linkage. A targeting sequence is present on the 5’ end of the conserved portion of the guide RNA. In certain embodiments, the targeting sequence is 20-25, preferably 22-24 nucleotides in length. In certain embodiments, the guide targeting sequence comprises on or more chemical modifications, for example modifications at one or more of nucleotides 1, 2, and 3, optionally all of nucleotides 1, 2, and 3 at the 5’ end of the guide RNA. In certain embodiments, the modification comprises a 2’-O-Me modification. In certain embodiments, the modification comprises a 2’-O-Me modification and a phosphorothioate linkage to the 3’ nucleotide, e.g., (mN*)3(N)17-22, preferably (mN*)3(N)21, wherein each of the nucleotides in the (N)21 portion is independently modified or unmodified. In certain embodiments, Exemplary SpyCas9 sgRNA-1, Exemplary NmeCas9 sgRNA-1, or an sgRNA, such as an sgRNA comprising an Exemplary SpyCas9 sgRNA-1, further includes a 3’ tail, e.g., a 3’ tail of 1, 2, 3, 4, or more nucleotides. In certain embodiments, the tail includes one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a 2’ deoxy (2’H-) modified nucleotide, an abasic nucleotide, a locked nucleic acid (LNA) nucleotide, an unlocked nucleic acid (UNA) nucleotide, or a phosphorothioate (PS) linkage between nucleotides, a terminal inverted abasic modified nucleotide; or a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. In certain embodiments, the modified nucleotide includes a PS linkage between nucleotides. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage between nucleotides. [0236] In certain embodiments, the hairpin region includes one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide; or a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. [0237] In certain embodiments, the upper stem region includes one or more modified nucleotides. In certain embodiments, the modified nucleotide selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide; or a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. [0238] In certain embodiments, the Exemplary SpyCas9 sgRNA-1 or the Exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a modified nucleotide. In certain embodiments, the modified nucleotide selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2- methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. [0239] In certain embodiments, the Exemplary SpyCas9 sgRNA-1 or the Exemplary NmeCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a sequence substituted nucleotide, wherein the pyrimidine is substituted for a purine. In certain embodiments, when the pyrimidine forms a Watson-Crick base pair in the single guide, the Watson-Crick based nucleotide of the sequence substituted pyrimidine nucleotide is substituted to maintain Watson-Crick base pairing. Linker containing gRNAs [0240] In certain embodiments, the gRNA comprises one or more internal linkers. As used herein, “internal linker” describes a non-nucleotide segment joining two nucleotides within a guide RNA. If the gRNA contains a spacer region, the internal linker is located outside of the spacer region (e.g., in the scaffold or conserved region of the gRNA). For Type V guides, it is understood that the last hairpin is the only hairpin in the structure, i.e., the repeat-anti-repeat region. The length of an internal linker may be dependent on, for example, the number of nucleotides replaced by the linker and the position of the linker in the gRNA. Internal linkers and their use in the context of gRNA are provided in WO2022261292. [0241] gRNAs disclosed herein may comprise an internal linker. In general, any internal linker compatible with the function of the gRNA may be used. It may be desirable for the linker to have a degree of flexibility. In some embodiments, the internal linker comprises at least two, three, four, five, six, or more on-pathway single bonds. A bond is on- pathway if it is part of the shortest path of bonds between the two nucleotides whose 5’ and 3’ positions are connected to the linker. [0242] As used herein the length of the internal linker can be defined by its bridging length. The “bridging length” of an internal linker as used herein refers to the distance or number of atoms in the shortest chain of atoms on the pathway from the first atom of the linker (bound to a 3’ substituent, such as an oxygen or phosphate, of the preceding nucleotide to the last atom of the linker (bound to a 5’ substituent, such as an oxygen or phosphate) of the following nucleotide) (e.g., from ~ to # in the structure of Formula (I) described below). Approximate predicted bridging lengths for various linkers are provided in a table below. [0243] Exemplary predicted linker lengths by number of atoms, number of ethylene glycol units, approximate linker length in Angstroms on the assumption that an ethylene glycol monomer is about 3.7 Angstroms, and suitable location for substitution of at least the entire loop portion of a hairpin structure are provided in the table 7 below. Substitution of two nucleotides requires a linker length of at least about 11 Angstroms. Substitution of at least 3 nucleotides requires a linker length of at least about 16 Angstroms. Table 7
Figure imgf000074_0001
[0244] In some embodiments, the internal linker comprises a structure of formula (I): ~-L0-L1-L2-# (I) wherein: ~ indicates a bond to a 3’ substituent of the preceding nucleotide; # indicates a bond to a 5’ substituent of the following nucleotide; L0 is null or C1-3 aliphatic; L1 is –[E1-(R1)]m-, where each R1 is independently a C1-5 aliphatic group, optionally substituted with 1 or 2 E2, each E1 and E2 are independently a hydrogen bond acceptor, or are each independently chosen from cyclic hydrocarbons, and heterocyclic hydrocarbons, and each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L2 is null, C1-3 aliphatic, or is a hydrogen bond acceptor. [0245] In some embodiments, L1 comprises one or more -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units (“ethylene glycol subunits”). In some embodiments, the number of -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units is in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. [0246] In some embodiments, m is 1, 2, 3, 4 or 5. In some embodiments, m is 1, 2, or 3. In some embodiments, m is 6, 7, 8, 9, or 10. [0247] In some embodiments, L0 is null. In some embodiments, L0 is -CH2- or - CH2CH2-. [0248] In some embodiments, L2 is null. In some embodiments, L2 is -O-, -S-, or C1-3 aliphatic. In some embodiments, L2 is -O-. In some embodiments, L2 is -S-. In some embodiments, L2 is -CH2- or -CH2CH2-. In the tables herein, L1 and L2, are optionally, C9 and C18, respectively as follows:
Figure imgf000075_0001
[0249] In certain embodiments, the internal linker has a bridging length of about 3-30 atoms, optionally 12-21 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA. In certain embodiments, the internal linker has a bridging length of about 6-18 atoms, optionally about 6-12 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA. In certain embodiments, the internal linker substitutes for 2-12 nucleotides. [0250] In some embodiments, the guide RNA comprises a nucleic acid sequence of SEQ ID NO: 268 or SEQ ID NO: 455, including modifications disclosed elsewhere herein. Table 8 shows various embodiments of the gRNA structures and species with possible number of internal linkers and positions. Table 8.
Figure imgf000076_0001
[0251] In certain embodiments, the internal linker is in a repeat-anti-repeat region of the gRNA. In certain embodiments, the internal linker substitutes for at least 4 nucleotides of the repeat-anti-repeat region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the repeat-anti-repeat region of a Spy Cas9 gRNA, corresponding to nucleotides 13-16 in SEQ ID NO: 268. In certain embodiments, the internal linker substitutes for the loop in the repeat-anti-repeat region of an Nme Cas9 gRNA, corresponding to nucleotides 49-52 in SEQ ID NO: 455. [0252] In certain embodiments, the internal linker substitutes for 2, 3, or 4 nucleotides of the nexus region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the nexus region of a Spy Cas9 gRNA corresponding to nucleotides 33-36 of SEQ ID NO: 268. [0253] In certain embodiments, the internal linker is in a hairpin region of the gRNA. In certain embodiments, the internal linker substitutes for at least 4 nucleotides of the hairpin region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the hairpin 1 region of a Spy Cas9 gRNA, corresponding to nucleotides 53-56 in SEQ ID NO: 268. In certain embodiments, the internal linker substitutes for the loop in the hairpin 1 region of an Nme Cas9 gRNA, corresponding to nucleotides 87-90 in SEQ ID NO: 455. In certain embodiments, the internal linker substitutes for at least 4 nucleotides the loop in the hairpin 2 region of an Nme Cas9 gRNA, corresponding to nucleotides 122-125 in SEQ ID NO: 455. In certain embodiments, the internal linker substitutes for the loop in the hairpin 1 region of an Nme Cas9 gRNA, corresponding to nucleotides 87-90 in SEQ ID NO: 455 and for at least 4 nucleotides the loop in the hairpin 2 region of an Nme Cas9 gRNA, corresponding to nucleotides 122-125 in SEQ ID NO: 455. Table 9. Exemplary SpyCas9 guide RNAs comprising linkers
Figure imgf000077_0001
Figure imgf000078_0002
Nucleotide modifications in modified sequences are indicated in Table 9 as follows: wherein “m” indicates a 2’-O-Me modification, a “*” indicates a phosphorothioate linkage between nucleotides, and within the individually indicated nucleotides, no modification indicates an RNA (2’-OH) with a phosphodiesterase backbone. (dS) denotes an abasic site having 1’,2’- dideoxyribose modification, (L3) denotes that the linker is S6, and (L4) denotes that the linker is S3. Table 10. Exemplary NmeCas9 guide RNAs comprising linkers
Figure imgf000078_0001
Figure imgf000079_0001
Nucleotide modifications in modified sequences are indicated in Table 10 as follows: wherein “m” indicates a 2’-O-Me modification, a “*” indicates a phosphorothioate linkage between nucleotides, and within the individually indicated nucleotides, no modification indicates an RNA (2’-OH) with a phosphodiesterase backbone. Even in the context of a modified sequence, each nucleotide of (N)20-25 is optionally independently modified. In certain examples, at least the first three nucleotides are modified, e.g., (mN*)3(N)17-22.
U G U G G U G A G A G G G A A A G A U A U U
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
[0254] In some embodiments, a composition comprising one or more guide RNAs (gRNAs) comprising targeting sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in the HBV genome are provided. In some embodiments, a liver cell comprising a genetic modification in an HBV sequence, e.g., an HBV sequence integrated into the endogenous genome of the cell, or as cccDNA in the cytoplasm, within the target sites of HBV is provided. In some embodiments, an engineered cell comprising a genetic modification in an HBV sequence is provided, wherein the genetic modification comprises a modification of at least one nucleotide within the genomic coordinates corresponding to an HBV targeting sequence selected from Tables 1A, 2A, 8, or 9, or that is complementary to at least 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides of nucleotides 163-466, 1-843, 1-1205, 2314- 3188, 1381-1846, or 1821-2460 of SEQ ID NO: 1. For example, 17, 18, preferably 19, or 20 contiguous nucleotides, or a complement thereof of nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621- 655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867- 1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891- 2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250- 269 of SEQ ID NO: 1, wherein the guide RNA is a Spy Cas9 guide RNA; or, for example, 20, 21, preferably 22, 23, or 24 contiguous nucleotides, or a complement thereof, of nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447- 470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390- 1413, 2951-2974, or 2958-2981; optionally nucleotides 447-470, 330-353, 755-778, or 713- 736 of SEQ ID NO: 1, wherein the guide RNA is an Nme Cas9 guide RNA. It is understood that there is substantial sequence variation among HBV genotypes, and that the sequence numbering provided for SEQ ID NO: 1 may not correspond to nucleotide sequence numbering of other HBV genome sequences. As used herein, a genomic modification of at least one nucleotide within the genomic coordinates of nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381-1846, or 1821-2460 of SEQ ID NO: 1, for example, nucleotides 57-76, 66- 85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574- 593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072- 2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; or, for example, nucleotides 35-58, 146-169, 153- 176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755- 778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 447-470, 330-353, 755- 778, or 713-736 of SEQ ID NO: 1, or corresponding to an HBV targeting sequence as provided in Table 1A, 2A, 12, or 14 is understood as at least one modification within a sequence that is at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 20 contiguous nucleotides or that is at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 24 contiguous nucleotides of the exemplary targeting sequences provided above. In some embodiments, an engineered cell comprising a genetic modification in an HBV sequence is provided, wherein the genetic modification comprises a modification of at least one nucleotide within a target site, i.e., within a nucleotide sequence at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 20 contiguous nucleotides or that is at least 80%, 85%, preferably 90% or 95% identical or complementary to at least 24 contiguous nucleotides of the exemplary target sites provided in Tables 1A and 2A, respectively. It is understood that the sequence of the HBV genome varies substantially across genotypes, and sometimes within individuals. Therefore, the specific target sites recited in Tables 1A and 2A may or may not be present in the HBV genome of a particular individual with an HBV infection. [0255] The guide RNA compositions provided herein are designed to recognize (e.g., hybridize to) a target sequence in an HBV gene, e.g., ORF. For example, the HBV target sequence may be recognized and cleaved, at one or both strands, by a provided Cas nuclease that is guided to the sequence by a guide RNA. In some embodiments, an RNA-guided DNA binding agent, such as a Cas nuclease, may be directed by a guide RNA to a target sequence of an HBV gene, where the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-guided DNA binding agent, such as a Cas nuclease, cleaves the target sequence on one or both strands. [0256] In some embodiments, the selection of the one or more guide RNAs is determined based on target sequences within an HBV gene. In some embodiments, one or more guide RNAs are targeted to nucleotides 163-466, 1-843, 1-1205, 2314-3188, 1381- 1846, or 1821-2460 of SEQ ID NO: 1. In some embodiments, one or more guide RNAs are targeted to nucleotides 163-466 of SEQ ID NO: 1. In some embodiments, the guide RNA is targeted to nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240- 259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629- 648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634- 2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1 wherein the guide RNA is a Spy Cas9 guide RNA. In some embodiments, the guide RNA is targeted to nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447- 470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1 wherein the guide RNA is an Nme Cas9 guide RNA. [0257] Without being bound by any particular theory, mutations (e.g., frameshift mutations resulting from indels, i.e., insertions or deletions, occurring as a result of a nuclease-mediated DSB, or sequence changes without a change in length, e.g., as a result of base editing) in certain regions of the gene may be less tolerable than mutations in other regions of the gene, thus the location of a mutation is an important factor in the amount or type of protein knockdown that may result. Similarly, without being bound by theory, the use of a base editor, e.g., a deaminase, to change the nucleotide sequence in certain regions of the gene may be less tolerable than mutations in other regions of the gene, resulting in disruption of a start codon, insertion of a stop codon, or a change in a coding sequence for an amino acid essential in protein structure or function, e.g., polymerase structure or function. In some embodiments, a gRNA complementary or having complementarity to a target sequence within HBV is used to direct the RNA-guided DNA binding agent to a particular location in the appropriate HBV gene. [0258] In some embodiments, the Spy guide sequence is at least 90% or 95%; or 100% identical to the reverse complement of a target sequence present in an HBV gene. In some embodiments, the target sequence is complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a Spy guide RNA and its corresponding target sequence is at least 80%, 85%, preferably at least 90%, or 95%; or 100%. In some embodiments, the target sequence and the guide sequence of the Spy gRNA may be 100% complementary or identical. [0259] In some embodiments, the target sequence and the guide sequence of the Spy gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides. [0260] In some embodiments, the Spy guide sequence comprises: (A) a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245- 264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630- 649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884- 1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269, of SEQ ID NO: 1; or (3) a target site selected from a target site of any one of HBV number S1-S93 in Table 1A, optionally a target site of any one of HBV number S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 in Table 1A; (B) a sequence identical to or complementary to 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, or to a target site provided in Table 1A, e.g., nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364- 383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697- 716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880- 1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982- 3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; (C) a sequence selected from the targeting sequence of G028550, G028551, G028552, G028553, G028554, G028555, G030415, G030418,G030423, G030424, G030426, G030428, G030429, G030430, G030431, G030432, G030435, G030436, G030437, G030439, G030446, G030454, G030456, G030457, G030459, G030460, G030464, G030465, G030468, G030469, G030470, G030471, G030474, G030477, G030478, G030481, G030484, G030485, G030487, G030488, G030489, G030490, G030491, G030492, G030493, G030494, G030496, G030497, G030498, or G030500, optionally G028550, G028554, or G028555; or (D) a sequence identical to or complementary to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1. [0261] In some embodiments, the Nme guide sequence is at least 90% or 95%; or 100% identical to the reverse complement of a target sequence present in an HBV gene. In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a targeting sequence of an Nme guide RNA and its corresponding target sequence is at least 80%, 85%, preferably at least 90%, or 95%; or 100%. In some embodiments, the target sequence and the targeting sequence of the gRNA are100% complementary or identical. [0262] In some embodiments, the target sequence and the targeting sequence of the Nme gRNA may contain at least one mismatch to the target sequence in the HBV genome. For example, the target sequence and the targeting sequence of the gRNA may contain 1 or 2, less preferably 3, or 4 mismatches, where the total length of the guide sequence is 24. In some embodiments, the target sequence and the targeting sequence of the gRNA may contain 1-2 mismatches where the length of the guide sequence is 24 nucleotides. [0263] In some embodiments, the Nme targeting sequence comprises: (A) a sequence at least 80%, 85%, preferably at least 90%, or 95% identical to or complementary to 24 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951- 2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1; or (3) a target site selected from any one of HBV number N1-N98 in Table 2A, optionally nucleotides of a target site of any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37in Table 2A; (B) a nucleic acid sequence identical to or complementary to 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460, for example nucleotides 35-58, 146- 169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755- 778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755- 778, or 713-736 of SEQ ID NO: 1; or of a target site provided in Table 2A, optionally nucleotides of a target site of any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; (C) a sequence selected from targeting sequence of G027224, G027225, G027228, G030107, G030108, G030115, G030120, G030123, G030125, G030130, G030134, G030138, G030158, G030162, G030167, G030180, or G030181; optionally G027224, G027225, G027228, G030107, G030115, G030120, G030123, G030125, G030130, G030162, or G030167; optionally G027224, G030107, G030115, or G030120; or (D) a sequence identical to or complementary to 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381- 1846, and nucleotides 1821-2460 of SEQ ID NO: 1. [0264] In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered. RNA-guided DNA binding agent; NmeCas9 [0265] RNA-guided DNA binding agents described herein encompass Neisseria meningitidis Cas9 (NmeCas9) and modified and variants thereof. In some embodiments, the NmeCas9 is Nme2 Cas9. In some embodiments, the NmeCas9 is Nme1 Cas9. In some embodiments, the NmeCas9 is Nme3 Cas9. [0266] Modified versions having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase RNA-guided DNA binding agent, such as a nickase Cas, e.g., a nickase Cas9, that induces a nick rather than a double strand break in the target DNA. [0267] In some embodiments, the NmeCas9 nuclease may be modified to contain only one functional nuclease domain. For example, the RNA-guided DNA binding agent may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. [0268] In some embodiments, a NmeCas9 nickase having a RuvC domain with reduced activity is used. In some embodiments, a NmeCas9 nickase having an inactive RuvC domain is used. In some embodiments, a NmeCas9 nickase having an HNH domain with reduced activity is used. In some embodiments, a NmeCas9 nickase having an inactive HNH domain is used. [0269] In some embodiments, a conserved amino acid within a NmeCas9 nuclease domain is substituted to reduce or alter nuclease activity. Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments, the Cas9 is capable of inducing a double strand break in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity. In some embodiments, a NmeCas9 may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include H588A (based on the N. meningitidis Cas9 protein). In some embodiments, the Cas protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include D16A (based on the NmeCas9 protein). [0270] In some embodiments, chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a NmeCas9 nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a NmeCas9 protein is a modified NmeCas9 nuclease. [0271] In some embodiments, the nuclease is modified to induce a point mutation or base change, e.g., through deamination. [0272] In some embodiments, the Cas protein comprises a fusion protein comprising a Cas nuclease (e.g., NmeCas9), which is a nickase or is catalytically inactive, linked to a heterologous functional domain. In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas nuclease (e.g., NmeCas9) linked to a heterologous functional domain (see, e.g., WO2014152432). In some embodiments, the catalytically inactive Cas9 is from the N. meningitidis Cas9. In some embodiments, the catalytically inactive Cas comprises mutations that inactivate the Cas. [0273] In some embodiments, the heterologous functional domain is a domain that modifies gene expression, histones, or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain. In some embodiments, the nuclease is a catalytically inactive Cas nuclease, such as dCas9. [0274] In some embodiments, the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. A heterologous functional domain such as a deaminase may be part of a fusion protein with a Cas nuclease having nickase activity or a Cas nuclease that is catalytically inactive discussed further below. [0275] In some embodiments, the Nme Cas9 has double stranded endonuclease activity. [0276] In some embodiments, the Nme Cas9 has nickase activity. [0277] In some embodiments, the Nme Cas9 comprises a dCas9 DNA binding domain. [0278] In some embodiments, the Nme Cas9 comprises an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 711, 716, 719, 722, or 730 (as shown in Table 40). In some embodiments, the Nme Cas9 comprises an amino acid sequence of any one of SEQ ID NOs: 711, 716, 719, 722, or 730 (as shown in Table 40). [0279] In some embodiments, the sequence encoding the NmeCas9 comprises a nucleotide sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 703, 704, 710, 715, 717-718, 720-721, or 728-729 (as shown in Table 40). In some embodiments, the sequence encoding the NmeCas9 comprises a nucleotide sequence of any one of SEQ ID NOs: 703, 704, 710, 715, 717-718, 720-721, or 728-729 (as shown in Table 40). [0280] In some embodiments, any of the foregoing levels of identity is at least 95%, 98%, or 99%; or 100%. [0281] Exemplary polynucleotides and compositions comprising a deaminase and an RNA-guided nickase are provided as SEQ ID NOs: 701, 704, 706, 707, 715-722, or 728-730 in Table 40 below. [0282] The RNA-guided DNA binding agent disclosed herein may further comprise a base-editing domain, such as a deaminase domain, that introduces a specific modification into a target nucleic acid. [0283] In some embodiments, a nucleic acid is provided that comprises an open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A), a C- terminal NmeCas9 nickase, and a first nuclear localization signal (NLS), wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI). [0284] In some embodiments, a second NLS is N-terminal to the NmeCas9 nickase. In some embodiments, the deaminase is N-terminal to an NLS (i.e., the first NLS or the second NLS). In some embodiments, the deaminase is N-terminal to all NLS in the polypeptide. In some embodiments, the deaminase is N-terminal to all NLS in the polypeptide, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI). [0285] In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, a polypeptide encoded by the mRNA is provided. [0286] In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase. In some embodiments, the polypeptide comprises, from N to C terminus, an optional NLS, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from N to C terminus, first and second NLSs, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase. In some embodiments, the polypeptide comprises, from N to C terminus, first and second NLSs, a cytidine deaminase (e.g., APOBEC3A), an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase. In some embodiments, the polypeptide comprises, from N to C terminus, A first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A NmeCas9 nickase. In some embodiments, the polypeptide comprises, from N to C terminus, A first NLS, a cytidine deaminase (e.g., APOBEC3A), a second NLS, an optional linker, and an Nme Cas9 nickase with an amino acid substitution in the HNH or HNH-like nuclease domain, e.g., a D16A Nme2Cas9 nickase. [0287] In some embodiments, the polypeptide comprising A3A and an RNA-guided nickase does not comprise a uracil glycosylase inhibitor (UGI). [0288] In some embodiments, a composition is provided comprising a first polypeptide, or an mRNA encoding a first polypeptide, comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI); and a second polypeptide, or an mRNA encoding a second polypeptide, comprising a uracil glycosylase inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide. [0289] In some embodiments, methods of modifying a target gene are provided comprising administering the compositions described herein. In some embodiments, the method comprises delivering to a cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), and a second nucleic acid comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid. [0290] In some embodiments, the methods comprise delivering to a cell a polypeptide comprising a deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal NmeCas9 nickase; a first nuclear localization signal (NLS); and a second NLS; wherein the first NLS and the second NLS are located to N-terminal to the sequence encoding the NmeCas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the polypeptide, and delivering to the cell a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the UGI. [0291] In some embodiments, a molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is from about 1:35 to from about 30:1. In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is not about 1:1. [0292] Similarly, in some embodiments, the molar ratio discussed above for the mRNA encoding the UGI protein to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase are similar if delivering protein. [0293] In some embodiments, the composition described herein further comprises at least one gRNA. In some embodiments, the composition described herein further comprises two gRNAs. In some embodiments, a composition is provided that comprises an mRNA described herein and at least one gRNA, e.g., two gRNAs. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is a dual guide RNA (dgRNA). [0294] In some embodiments, the composition is capable of effecting genome editing upon administration to the subject. Cytidine deaminase; APOBEC3A Deaminase [0295] Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol.22:367- 77, 2005; Conticello, Genome Biol.9:229, 2008; Muramatsu et al., J. Biol. Chem.274: 18470-6, 1999); and Carrington et al., Cells 9:1690 (2020)). [0296] In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is an APOBEC3A deaminase (A3A).In some embodiments, the deaminase comprises an APOBEC3A deaminase. [0297] In some embodiments, an APOBEC3A deaminase (A3A) disclosed herein is a human A3A. In some embodiments, an APOBEC3A deaminase (A3A) disclosed herein is a human A3A. In some embodiments, the A3A is a wild-type A3A. [0298] In some embodiment, the A3A is an A3A variant. A3A variants share homology to wild-type A3A, or a fragment thereof. In some embodiments, a A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to a wild type A3A. In some embodiments, the A3A variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type A3A. In some embodiments, the A3A variant comprises a fragment of an A3A, such that the fragment has at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of a wild-type A3A. [0299] In some embodiments, an A3A variant is a protein having a sequence that differs from a wild-type A3A protein by one or several mutations, such as substitutions, deletions, insertions, one or several single point substitutions. In some embodiments, a shortened A3A sequence could be used, e.g. by deleting N-terminal, C-terminal, or internal amino acids. In some embodiments, a shortened A3A sequence is used where one to four amino acids at the C-terminus of the sequence is deleted. In some embodiments, an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild- type sequence). [0300] In some embodiments, the wild-type A3A is a human A3A (UniPROT accession ID: p31941, SEQ ID NO: 734). [0301] In some embodiments, the A3A disclosed herein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 734. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the A3A comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 734. In some embodiments, the A3A comprises an amino acid sequence with at least 99% identity to A3A ID NO: 734. In some embodiments, the A3A comprises the amino acid sequence of SEQ ID NO: 734. [0302] In some embodiments, the cytidine deaminase disclosed herein comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 734-216735. In some embodiments, any of the foregoing levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 709 or 735. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 709 or 735. Linkers [0303] In some embodiments, the polypeptide comprising the deaminase and the RNA-guided nickase described herein further comprises a linker that connects the deaminase and the RNA-guided nickase. In some embodiments, the linker is a peptide linker. In some embodiments, the nucleic acid encoding the polypeptide comprising the deaminase and the RNA-guided nickase further comprises a sequence encoding the peptide linker. In some embodiments, mRNAs encoding the deaminase-linker-RNA-guided nickase fusion protein are provided. [0304] In some embodiments, the peptide linker is any stretch of amino acids having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids. [0305] In some embodiments, the peptide linker is the 16 residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 652), SGSETPGTSESA (SEQ ID NO: 653), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 654). [0306] In some embodiments, the peptide linker comprises a (GGGGS)n (SEQ ID NO: 655), a (G)n (SEQ ID NO: 823), an (EAAAK)n(SEQ ID NO: 656), a (GGS)n (SEQ ID NO: 824), an SGSETPGTSESATPES (SEQ ID NO: 657) motif (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol.2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP)n motif (SEQ ID NO: 825), or a combination of any of these, wherein n is independently an integer between 1 and 30. See, WO2015089406, e.g., paragraph [0012], the entire content of which is incorporated herein by reference. [0307] In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NOs: 652-657. Modified gRNAs and mRNAs [0308] In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); and (iv) modification of the 3' end or 5' end of the oligonucleotide to provide exonuclease stability, e.g., with 2’ O-me, 2’ halide, or 2’ deoxy substituted ribose; or inverted abasic terminal nucleotide, or replacement of phosphodiester with phosphorothioate. [0309] Chemical modifications such as those listed above can be combined to provide modified gRNAs or mRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In certain embodiments, up to 15% of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA. [0310] In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, 10%, 15%, preferably at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the positions in a modified gRNA are modified nucleosides or nucleotides. In some embodiments, at least 5% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments, at least 10% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments at least 15% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments preferably at least 20% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments, no more than 65% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 55% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 50% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 10-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-50% of the positions in the modified gRNA are modified nucleotides and the nuclease is a Spy Cas9 nuclease. In some embodiments, range 30-70% of the positions in the modified gRNA are modified nucleotides and the nuclease is an Nme Cas9 nuclease. [0311] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. [0312] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. [0313] Examples of modified phosphate groups include, phosphorothioate, borano phosphate esters, methyl phosphonates, phosphoroamidates, phosphodithioate, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. [0314] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications, e.g., an amide linkage. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, carboxymethyl, carbamate, amide, thioether. Further examples of moieties which can replace the phosphate group can include, without limitation, e.g., ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. [0315] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. [0316] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'- alkoxide ion. [0317] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).2' modifications can include hydrogen (i.e. deoxyribose sugars); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein. [0318] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides. As used herein, a single abasic sugar is not understood to result in a discontinuity of a duplex. [0319] In certain embodiments, 2’ modifications, include, for example, modifications include 2’-OMe, 2’-F, 2’-H, optionally 2’-O-Me. [0320] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uridine (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base. [0321] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or internal nucleosides may be modified, or the sgRNA may be chemically modified throughout. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification. Certain embodiments comprise a 5’ end modification and a 3’ end modification. [0322] In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018/107028, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2017/136794, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2019/237069, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2021/119275, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in Nme guides, the contents of which are hereby incorporated by reference in their entirety. [0323] In some embodiments, the sgRNA comprises any of the modification patterns shown herein, where N is any natural or non-natural nucleotide, and wherein the totality of the N’s comprise an HBV guide sequence as described herein in Table 1A. In some embodiments, the modified sgRNA comprises a sequence shown in Table 15 or in Table 9. Table 15: Modified Spy Guide RNA Sequences
Figure imgf000132_0001
Figure imgf000133_0001
wherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH), wherein the totality of N’s comprise a targeting sequence comprising (A) a sequence at least 80%, 85%, preferably 90% or 95% identical to or complementary to at least 20 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381- 1846, or nucleotides 1821-2460 of SEQ ID NO: 1; (2) nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356- 392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694- 716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073- 2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; or (3) a target site selected from a target site of any one of HBV number S1-S93 in Table 1A, optionally a target site of any one of HBV number S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 in Table 1A; (B) a sequence identical to or complementary to 17, 18, preferably 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821- 2460; for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373- 392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713- 732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883- 1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1, or to a target site provided in Table 1A; (C) a sequence selected from the targeting sequence of G028550, G028551, G028552, G028553, G028554, G028555, G030415, G030418,G030423, G030424, G030426, G030428, G030429, G030430, G030431, G030432, G030435, G030436, G030437, G030439, G030446, G030454, G030456, G030457, G030459, G030460, G030464, G030465, G030468, G030469, G030470, G030471, G030474, G030477, G030478, G030481, G030484, G030485, G030487, G030488, G030489, G030490, G030491, G030492, G030493, G030494, G030496, G030497, G030498, or G030500, optionally G028550, G028554, or G028555; or (D) a sequence identical to or complementary to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1- 843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; or (2) nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361- 380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695- 714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299- 2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1. For example, where the N’s are replaced with any of the guide sequences disclosed herein in Table 1A. In certain embodiments, when the totality of N’s comprise a guide sequence, within N17, each N of the N17 may be independently modified, e.g., modified with a 2’-OMe modification. [0324] In some embodiments, the sgRNA comprises a sequence wherein the totality of the N’s comprise an HBV targeting sequence as described herein in Table 2A. In some embodiments, the modified sgRNA comprises a sequence provided in Table Table 16 or Table 10: Table 16: Exemplary Modified Nme Guide RNA Motifs
Figure imgf000135_0001
wherein “m” indicates a 2’-O-Me modification, and a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and a phosphorothioate linkage. A targeting sequence is present on the 5’ end of the conserved portion of the guide RNA. In certain embodiments, the targeting sequence is 20-25, preferably 22-24 nucleotides in length. In certain embodiments, the guide targeting sequence comprises on or more chemical modifications, for example modifications at one or more of nucleotides 1, 2, and 3, optionally all of nucleotides 1, 2, and 3 at the 5’ end of the guide RNA. In certain embodiments, the modification comprises a 2’-O- Me modification. In certain embodiments, the modification comprises a 2’-O-Me modification and a phosphorothioate linkage to the 3’ nucleotide, e.g., (mN*)3(N)17-22, preferably (mN*)3(N)21, wherein each of the nucleotides in the (N)21 portion is independently modified or unmodified. In certain embodiments, the (N)20-25 has the following sequence and modification pattern mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN. [0325] In certain embodiment, the totality of N’s comprise an HBV targeting sequence comprising: (A) a sequence at least 80%, 85%, preferably at least 90%, or 95% identical, or 100% identical to or complementary to 24 contiguous nucleotides of: (1) nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460of SEQ ID NO: 1; (2) nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713- 736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1; or (3) a target site selected from any one of HBV number N1-N98 in Table 2A, optionally nucleotides of a target site of any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; (B) a nucleic acid sequence identical to or complementary to 21, 22, 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981; optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1; or of a target site provided in Table 2A; (C) a sequence selected from targeting sequence of G027224, G027225, G027228, G030107, G030108, G030115, G030120, G030123, G030125, G030130, G030134, G030138, G030158, G030162, G030167, G030180, or G030181; optionally G027224, G027225, G027228, G030107, G030115, G030120, G030123, G030125, G030130, G030162, or G030167; optionally G027224, G030107, G030115, or G030120; or (D) a sequence identical to or complementary to 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381- 1846, and nucleotides 1821-2460 of SEQ ID NO: 1; for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740- 763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755- 778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755- 778, or 713-736 of SEQ ID NO: 1; or of a target site provided in Table 2A. For example, where the N’s are replaced with any of the guide sequences disclosed herein in Table 2A. In certain embodiments, when the totality of N’s comprise a guide sequence, within (N)20-25, each N of the (N)20-25 may be independently modified, e.g., modified with a 2’-OMe modification, optionally further with a PS modification, particularly at 1, 2, or 3 terminal nucleotides. In certain embodiments, the (N)20-25 has the following sequence and modification pattern mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN. [0326] Any of the modifications described below may be present in the gRNAs and mRNAs described herein. [0327] In the context of chemically modified sequences, “A,” “C,” “G,” “N,” and “U” denote an RNA nucleotide, i.e., 2’-OH with a phosphodiesterase linkage to the 3’ nucleotide. [0328] The terms “mA,” “mC,” “mU,” or “mG” are used to denote an adenine, cytosine, uridine, or guanidine nucleotide, respectively, that has been modified with 2’-O-Me. [0329] Modification with 2’-O-methyl can be depicted as follows:
Figure imgf000137_0001
[0330] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. [0331] In this application, the terms “fA,” “fC,” “fU,” or “fG” are used to denote a nucleotide that has been substituted with 2’-F. [0332] Substitution of 2’-F can be depicted as follows:
Figure imgf000138_0001
[0333] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one non-bridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos. [0334] A “*” is used to denote a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a PS bond. [0335] In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” are used to denote a nucleotide that has been substituted with 2’-O-Me and that is linked to the next (e.g., 3’) nucleotide with a PS bond.
[0336] The diagram below shows the substitution of S- into a non-bridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:
Figure imgf000139_0001
[0337] Abasic nucleotides refer to those which lack nitrogenous bases. The diagram below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base. As used herein, the presence of a single abasic site is not considered to disrupt a duplex, e.g., a duplex formed between the targeting sequence of a guide RNA and a target site in the genome:
Figure imgf000139_0002
[0338] Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage). Such inverted bases can only be present as a terminal nucleotide. In chemical synthesis methods performed 3’ to 5’, inverted bases do not have 5’ hydroxy available to grow the chain. For example:
Figure imgf000140_0001
[0339] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap. [0340] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2’-O-Me, 2’-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability or performance. [0341] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds. [0342] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise an inverted abasic nucleotide. [0343] In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in Table 4, for example mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCG GmU*mG*mC*mU (SEQ ID NO: 669) where each A, C, G, U, and N is an RNA nucleotide, 2’-OH and phosphodiester linkage to the 3’ nucleotide, m indicates a 2'-O-methyl (2'-O-Me) modified nucleotide, and * indicates a phosphorothioate linkage between nucleotides , and where the totality of the N’s comprise a guide sequence that directs a nuclease to a target sequence in HBV, e.g., the target sequences shown in Table 1A. In certain embodiments, the guide sequence comprises a sequence of any one of SEQ ID NOs: 2-94 shown in Table 1A. [0344] In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in Table 6, for example [0345] mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN mGUUGmUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAU*AAGmG mCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUm CmUGGCAUCG*mU*mU (SEQ ID NO: 816) [0346] mN*mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNNmGUUG mUmAmGmCUCCCmUmGmAmAmAmCmCGUUmGmCUAmCAAUAAGmGmCCmGm UmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGmCCmUmUmCmUGGC AUCG*mU*mU (SEQ ID NO: 817) [0347] mN *mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN mGUUGmUmAmGmCUCCCmUmUmCmGmAmAmAmGmAmCmCGUUmGmCUAmCA AU*AAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmG mCCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 818) [0348] mN *mN*mN*mNmNNNmNmNNmNNmNNNNNmNNNNmNNN mGUUGmUmAmGmCUCCCmUmUmCmGmAmAmAmGmAmCmCGUUmGmCUAmCA AUAAGmGmCCmGmUmCmGmAmAmAmGmAmUGUGCmCGmCAAmCGCUCUmGm CCmUmUmCmUGGCAUCG*mU*mU (SEQ ID NO: 819) [0349] where each A, C, G, U, and N is an RNA nucleotide, 2’-OH and phosphodiester linkage to the 3’ nucleotide, m indicates a 2'-O-methyl (2'-O-Me) modified nucleotide, and * indicates a phosphorothioate linkage between nucleotides, and where the totality of the N’s comprise a guide sequence that directs a nuclease to a target sequence in HBV, e.g., the target sequences shown in Table 2A. In certain embodiments, the guide sequence comprises a sequence of any one of SEQ ID NOs: 95-192 shown in Table 2A. [0350] As noted above, in some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease, as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease, is provided, used, or administered. In some embodiments, the ORF encoding an RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified. [0351] In some embodiments, the mRNA or modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl- pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5- methoxyuridine. [0352] In some embodiments, an mRNA disclosed herein comprises a 5’ cap, such as a Cap0, Cap1, or Cap2. A 5’ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5’- triphosphate to the 5’ position of the first nucleotide of the 5’-to-3’ chain of the mRNA, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2’-methoxy and a 2’-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA. [0353] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7- methylguanine 3’-methoxy-5’-triphosphate linked to the 5’ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap in which the 2’ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘anti- reverse’ cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG,” RNA 7: 1486–1495. The ARCA structure is shown below.
Figure imgf000143_0001
[0354] CleanCapTM AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCapTM GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally.3’-O-methylated versions of CleanCapTM AG and CleanCapTM GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively, or CleanCap AU: TriLink Biotechnologies as Cat. Nos. N-7114. The CleanCapTM AG structure is shown below.
Figure imgf000143_0002
[0355] Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem.269, 24472-24479. [0356] In some embodiments, the mRNA further comprises a poly-adenylated (poly- A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some embodiments, the poly-A tail includes non-adenine nucleotides, i.e., is an interrupted poly-A tail. In certain embodiments, the poly-A tail is interrupted by a non-adenine nucleotide about every 40, 50, 60, 70, 80, or 90 nucleotides. In certain embodiments, the poly-A tail is interrupted by a non- adenine nucleotide about every 50 nucleotides. Ribonucleoprotein complex [0357] In some embodiments, a composition is encompassed comprising one or more sgRNAs comprising one or more guide sequences from Table 1A or one or more sgRNAs from Table 2A and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9. In some embodiments, the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, Neisseria meningitidis, and other prokaryotes as known in the art , and modified (e.g., engineered or mutant) versions thereof. [0358] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. [0359] In some embodiments, the gRNA together with an RNA-guided DNA binding agent is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the gRNA together with a Cas nuclease is called a Cas RNP. In some embodiments, the RNP comprises Type-I, Type-II, or Type-III components. In some embodiments, the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP. [0360] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA. [0361] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas nuclease may be a modified nuclease. [0362] In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity. [0363] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., US Pat. No.8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. [0364] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain. [0365] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or RuvC or RuvC-like domains for N. meningitidis include Nme2Cas9D16A (HNH nickase) and Nme2Cas9H588A (RuvC nickase) [0366] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA. [0367] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 20140186958; US 20150166980; and US 20190338308. [0368] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide). [0369] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, or 4 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. In some embodiments, the NLS is not linked to the C-terminus. It may also be inserted within the RNA-guided DNA binding agent sequence. In certain circumstances, at least the two NLSs are the same (e.g., two SV40 NLSs). In certain embodiments, at least two different NLSs are present the RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. [0370] SV40 NLS, PKKKRKV (SEQ ID NO: 679) or PKKKRRV (SEQ ID NO: 680). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 681). In some embodiments, the NLS sequence may comprise LAAKRSRTT (SEQ ID NO: 682), QAAKRSRTT (SEQ ID NO: 683), PAPAKRERTT (SEQ ID NO: 684), QAAKRPRTT (SEQ ID NO: 685), RAAKRPRTT (SEQ ID NO: 686), AAAKRSWSMAA (SEQ ID NO: 687), AAAKRVWSMAF (SEQ ID NO: 688), AAAKRSWSMAF (SEQ ID NO: 689), AAAKRKYFAA (SEQ ID NO: 690), RAAKRKAFAA (SEQ ID NO: 691), or RAAKRKYFAV (SEQ ID NO: 692). The NLS may be a snurportin-1 importin- (IBB domain, e.g. an SPN1-imp sequence. See Huber et al., 2002, J. Cell Bio., 156, 467-479. In a specific embodiment, a single PKKKRKV (SEQ ID NO: 693). In some embodiments, the first and second NLS are independently selected from an SV40 NLS, a nucleoplasmin NLS, a bipartite NLS, a c-myc like NLS, and an NLS comprising the sequence KTRAD (SEQ ID NO: 820). In certain embodiments, the first and second NLSs may be the same (e.g., two SV40 NLSs). In certain embodiments, the first and second NLSs may be different. [0371] In some embodiments, the first NLS is a SV40NLS and the second NLS is a nucleoplasmin NLS. [0372] In some embodiments, the SV40 NLS comprises a sequence of PKKKRKVE (SEQ ID NO: 694) or KKKRKVE (SEQ ID NO: 695). In some embodiments, the nucleoplasmin NLS comprises a sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 696). In some embodiments, the bipartite NLS comprises a sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 697). In some embodiments, the c-myc like NLS comprises a sequence of PAAKKKKLD (SEQ ID NO: 698). [0373] One or more linkers are optionally included at the fusion site of the NLS to the nuclease, or between NLS when more than one is present. [0374] In some embodiments, one or more NLS(s) according to any of the foregoing embodiments are present in the RNA-guided DNA-binding agent in combination with one or more additional heterologous functional domains. One or more linkers are optionally included at the fusion site. [0375] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal- precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5). [0376] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1 ), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira- Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis (SEQ ID NO: 821), 8xHis (SEQ ID NO: 822), biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins. [0377] In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No.9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol.31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA. In some embodiments, the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. Determination of efficacy of gRNAs [0378] In some embodiments, the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the gRNA is expressed together with an RNA-guided DNA binding agent, such as a Cas protein, e.g. Cas9. In some embodiments, the gRNA is delivered to or expressed in a cell line that already stably expresses an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase. In some embodiments the gRNA is delivered to a cell as part of a RNP. In some embodiments, the gRNA is delivered to a cell along with a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g. Cas9 nuclease or nickase. [0379] As described herein, use of an RNA-guided DNA nuclease and a guide RNA disclosed herein can lead to double-stranded breaks in the DNA which can produce errors in the form of insertion/deletion (indel) mutations upon repair by cellular machinery. Many mutations due to indels alter the reading frame or introduce premature stop codons and, therefore, produce a non-functional protein. In other embodiments, use of an RNA-guided DNA nuclease results in a base change that results in a premature stop codon, disruption of a start codon or regulatory sequence, or introduces a mutation that disrupts function of the encoded protein, or a combination thereof. In some embodiments, the efficacy of particular gRNAs is determined based on in vitro models. In some embodiments, the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9, e.g., available from ATCC). In certain embodiments, the cell line is a HepAD38 cell line. In some embodiments, the in vitro model is a primary cell line, e.g., a primary liver cell line, e.g., primary hepatocytes. In some embodiments the in vitro model is a peripheral blood mononuclear cell (PBMC). In some embodiments, the in vitro model is a T cell, such as primary human T cells. With respect to using primary cells, commercially available primary cells can be used to provide greater consistency between experiments. In some embodiments, the number of off-target sites at which a deletion or insertion occurs in an in vitro model (e.g., in a primary hepatocyte, in a T cell) is determined, e.g., by analyzing genomic DNA from transfected cells in vitro with Cas9 mRNA and the guide RNA. In some embodiments, such a determination comprises analyzing genomic DNA from the cells transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide. Exemplary procedures for such determinations are provided in the working examples in which various cell types are used. [0380] In some embodiments, the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process. In some embodiments, a cell line comparison of data with selected gRNAs is performed. In some embodiments, cross screening in multiple cell models is performed. [0381] In some embodiments, the efficacy of a guide RNA is measured by percent indels or percent genetic modifications of the HBV genome sequence, either integrated into the genome of the host cell or as cccDNA in the cytoplasm. In some embodiments, the efficacy of a guide RNA is measured by percent indels or percent genetic modifications at an HBV genome locus, e.g., HepAD38 (Genotype D) ( see, e.g., Ladner SK et al.1997, AAC 41:1715–1720). In some embodiments, the efficacy of a guide RNA is measured by percent indels or percent genetic modifications of an HBV target site of Tables 1A and 2A. In some embodiments, the percent editing of an HBV genome locus compared to the percent indels or genetic modifications necessary to achieve reduction, e.g., knockdown, of the HBV protein products. In some embodiments, the efficacy of a guide RNA is measured by reduced expression of an HBV protein. In embodiments, said reduced expression of a protein is as measured by ELISA, e.g., as described herein. In certain embodiments, the protein is a secreted protein, e.g., in cell culture media or in blood or a fraction derived therefrom, e.g., serum. [0382] In some embodiments, the HBV protein expression is reduced using the methods and compositions disclosed herein. In some embodiments, the level of protein as determined, e.g., by ELISA, is reduced by at least 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, or 99% relative to a control population of unmodified cells. In some embodiments, the level of protein is determined by a protein activity assay, e.g., a polymerase assay. [0383] An “unmodified cell” (or “unmodified cells”) refers to a control cell (or cells) of the same type of cell in an experiment or test, wherein the “unmodified” control cell has not been contacted with an HBV guide. Therefore, an unmodified cell (or cells) may be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that does not target HBV. As used herein, an “unmodified cell” can include the HepAD38 cell line that has not been contacted with an HBV guide. [0384] In some embodiments, the efficacy of a guide RNA is measured by the number or frequency of indels or genetic modifications at off-target sequences within the genome of the target cell type, such as a primary hepatocyte cell. In some embodiments, efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g., <5%) in a cell population or relative to the frequency of indel creation at the target site. Thus, the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., a primary hepatocyte cell), or which produce a frequency of off-target indel formation of <5% in a cell population or relative to the frequency of indel creation at the target site. In some embodiments, the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., primary hepatocyte cell) as compared to a control cell. In some embodiments, guide RNAs are provided which produce indels at less than 5 validated off-target sites. In some embodiments, guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 validated off-target site(s). In some embodiments, the off-target site(s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome. [0385] In some embodiments, the efficacy of a guide RNA is measured in vivo, e.g., in an animal or animal model having a DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, i.e., having a DNA sequence sufficiently complementary to the targeting sequence in the guide RNA proximal to a cognate PAM for the guide and nuclease. In certain embodiments, the animal has an endogenous DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA. In certain embodiments, the animal model is a transgenic model, e.g., a mouse model having an inserted DNA sequence susceptible to cleavage by a nuclease targeted by the guide RNA, e.g., a mouse having an inserted DNA sequence, e.g., a viral DNA sequence or a human DNA sequence, e.g. a sequence from a human albumin. The inserted sequence may or may not include one or more intron sequence or regulatory sequence, e.g., 3’ UTR, 5’ UTR, present in the human gene in its native context. In certain embodiments, the human DNA sequence may replace the homologous endogenous DNA sequence, the mouse albumin gene is replaced by the human albumin gene. In certain embodiments, the human gene is present in the mouse in the context of a human hepatocyte, e.g., a mouse with a humanized liver, e.g., as available from PhoenixBio or Inotiv. In certain embodiments, the inserted DNA sequence is a viral DNA sequence in a human hepatocyte in a mouse with a humanized liver. In certain embodiments, the inserted DNA sequence is a viral sequence containing the target site inserted into a viral vector of a different virus, e.g., an HBV DNA sequence in an AAV vector. [0386] In some embodiments, detecting gene editing events, such as the formation of insertion/deletion (“indel”) mutations and insertion or homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (herein after referred to as “LAM-PCR,” or “Linear Amplification (LA)” method). In some embodiments, detecting gene editing events that are base editing events, e.g., changes in nucleotide sequence without insertion or deletion of nucleotides, are detected by next generation sequencing (NGS). In some embodiments, the efficacy of a guide RNA is measured by the levels of functional protein or protein complexes comprising the expressed protein product of the gene, e.g., polymerase activity assay. In some embodiments, the efficacy of a guide RNA is measured by ELISA. Genetic modification for inhibition of target gene expression [0387] The cells or population of cells comprise a genetic modification, e.g., of a nucleic acid sequence encoding an HBV gene, e.g., integrated into the host genome or as cccDNA present in the cytoplasm. [0388] In some embodiments, the engineered cells or population of cells comprise a genetic modification of an HBV gene as assessed by sequencing, e.g., NGS, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90% 95%, or 99% of cells comprise an insertion, deletion, or substitution in the HBV sequence. It is understood that there are multiple HBV genotypes, and that genetic modifications are compared to the HBV sequence present in the cell which may or may not be different from the HBV sequence, SEQ ID NO: 1, provided herein. In some embodiments, at least 50% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 55% of cells in the population comprise a modification. In some embodiments, at least 60% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 65% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. on selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 70% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 75% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 80% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 85% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some preferred embodiments, at least 90% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 95% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. In some embodiments, at least 99% of cells in the population comprise a modification selected from an insertion, a deletion, and a substitution in the HBV sequence. [0389] In some embodiments, HBV protein expression, e.g., HBsAg protein expression, is decreased by at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90%, 95%, or 99% as compared to a level wherein the HBV gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 70%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene, has not been modified. In some embodiments, expression of HBsAg is decreased by at least 75%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 80%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 85%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 90%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by at least 95%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. In some embodiments, expression of HBsAg is decreased by no more than 99%, as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HBs gene has not been modified. Assays for HBsAg protein and mRNA expression are known in the art. [0390] In some embodiments, HBV protein expression, e.g., HB pol expression as demonstrated by protein level or activity, is decreased by at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90%, 95%, or 99% as compared to a level wherein the HBV gene has not been modified. In some embodiments, expression of HB P is decreased by at least 70%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene, has not been modified. In some embodiments, expression of HB P is decreased by at least 75%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is decreased by at least 80%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified. In some embodiments, expression of HB P is decreased by at least 85%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is preferably decreased by at least 90%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified. In some embodiments, expression of HB P is decreased by at least 95%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene, has not been modified. In some embodiments, expression of HB P is decreased by at least 99%, or to below the limit of detection of the assay as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P gene has not been modified. In some embodiments, expression of HB P is decreased by no more than 95%, as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified. In some embodiments, expression of HB P is decreased by no more than 99%, as compared to a suitable control, e.g., wherein an HBV gene, e.g., the HB P has not been modified. Assays for HB P protein expression and activity, and mRNA expression are known in the art. [0391] In some embodiments, HBV protein expression, e.g., HBsAg expression, e.g., as demonstrated by HBsAg level, and HB P expression, e.g., as demonstrated by protein level or activity, are both decreased by at least 50%, 55%, 60%, 65%, 70%, 75%, preferably at least 80%, 85%, more preferably at least 90%, 95%, or 99% as compared to a level wherein the HBV gene has not been modified. In some embodiments, HBsAg expression and HB P expression are both reduced by use of one gRNA targeted to a single site in within overlapping region of the HBsAg and HB P ORFs, e.g., within the HBsAg ORF and the HB P ORF in SEQ ID NO: 1. It is understood that due to the variation in HBV genomes, a gRNA that is effective in targeting both the HBsAg ORF and the HB P ORF in SEQ ID NO: 1 may not be useful in targeting both ORFs in all HBV genotypes. [0392] In some embodiments, HBsAg expression and HB pol expression are reduced by use of at least two gRNA targeted to distinct sites, e.g., non-overlapping sites, in the HBsAg ORF and HB pol ORFs. [0393] It is understood that in some embodiments, in vivo, the level of expression may be inhibited in one, but not in all, tissues where the target gene is expressed. In the case of HBV, the level of expression of an HBV protein may be inhibited in one, but not all, HBV genotypes in which the ORFs are expressed. In certain embodiments, surrogate markers can be used to monitor changes in expression. For example, many proteins made in the liver are secreted into circulation; therefore, the level of inhibition of expression may be determined by or correlated to a decrease in levels in protein in the blood. In certain embodiments, inhibition of expression in the liver can result in a change in a metabolite or other biomarker in a body fluid, e.g., blood or urine. The change in the level of the metabolite can be correlated with the level of inhibition of expression. Such correlations can be useful for monitoring the level of inhibition of expression as serial biopsies, e.g., serial liver biopsies, are not practical for monitoring in human subjects, or often in animal models. [0394] In some embodiments, the target gene is genetically modified resulting in inhibition of expression in a cell using a guide RNA with an RNA-guided DNA binding agent. In some embodiments, disclosed herein are cells engineered by inducing a break (e.g., double-stranded break (DSB) or single-stranded break (nick)) within the target genes of a cell, e.g., using a guide RNA with an RNA-guided DNA-binding agent (e.g., a CRISPR/Cas system). The methods may be used in vitro, e.g., for screening guides, or in vivo, e.g., to provide a therapeutic benefit. [0395] In some embodiments, the guide RNAs mediate a target-specific cutting of one or both DNA strands by an RNA-guided DNA-binding agent (e.g., Cas nuclease) at a site described herein within a target gene. It will be appreciated that, in some embodiments, the guide RNAs comprise guide sequences that bind to, or are capable of binding to, those regions. IV. Methods and Uses Including Therapeutic Methods and Uses of Genome Editing Agents [0396] The gRNAs and associated methods and compositions disclosed herein are useful for making genome editing therapeutic agents. [0397] In some embodiments, the gRNAs comprising the guide sequences of Tables 1A and 2A together with an RNA-guided DNA nuclease such as a Cas nuclease induce breaks, either single stranded breaks or double stranded breaks. Double stranded breaks lead to non-homologous ending joining (NHEJ) during repair, leading to a modification, e.g., a mutation, in an HBV gene. In some embodiments, NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in an HBV gene. Single stranded breaks can be coupled to base editing resulting in a sequence change, e.g., generating a stop codon, disrupting a start site, or a mutation that disrupts function of the ORF. In certain embodiments, gRNAs comprising guide sequences targeted to target genomic sequences are also delivered to the cell together with RNA-guided DNA nuclease such as a Cas nuclease, either together or separately, to make a genetic modification in a target genomic sequence to inhibit the expression of a full-length expression product from the target gene. Due to the overlapping nature of the HBV genome, one guide RNA may cause changes in more than one ORF in the HBV genome. In certain embodiments, the gRNAs are sgRNAs. [0398] In some embodiments, the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse in which the liver has been repopulated with human hepatocytes. [0399] In some embodiments, the guide RNAs, compositions, and formulations are used to produce a cell in vivo, e.g., liver cell, e.g., a hepatocyte with a genetic modification in an HBV gene, that may be integrated into the host genome or present as an episomal copy, e.g., as cccDNA. Delivery of gRNA Compositions [0400] Lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs and compositions disclosed herein in vivo and in vitro. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein. [0401] In some embodiments, a method for delivering any one of the cells or populations of cells disclosed herein to a subject is provided, wherein the gRNA is delivered via an LNP in vivo. In some embodiments, the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9. [0402] In some embodiments, a composition comprising any one of the gRNAs disclosed and an LNP is provided. In some embodiments, the composition further comprises a Cas9 or an mRNA encoding Cas9. In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI) or an mRNA encoding a UGI. [0403] In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder. [0404] In some embodiments, a method for delivering any one of the gRNAs disclosed herein in vivo is provided, wherein the gRNA is associated with an LNP. In some embodiment, the gRNA is not associated with an LNP. In some embodiments, the gRNA/LNP or gRNA is also associated with a Cas9 or an mRNA encoding Cas9. In some embodiments, the gRNA/LNP, gRNA, Cas9, or mRNA encoding a Cas9, is also associated with a UGI or an mRNA encoding a UGI. [0405] In some embodiments, the guide RNA compositions described herein, alone or encoded on one or more vectors, are formulated in or administered via a lipid nanoparticle; see e.g., WO2017/173054 and WO2021/222287, the contents of each of which are hereby incorporated by reference in their entirety. [0406] In certain embodiments, DNA or RNA vectors encoding any of the guide RNAs comprising any one or more of the guide sequences described herein are provided. In some embodiments, in addition to guide RNA sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9. In one embodiment, the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In one embodiment, the Cas9 is from Neisseria meningitidis (i.e., Nme Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA. [0407] In some embodiments, the components can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or they can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus). Methods and compositions for non-viral delivery of nucleic acids include lipofection, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acid (e.g., naked DNA/RNA), artificial virions, and agent-enhanced uptake of DNA. Methods of delivery appropriate for in vitro screening may not be appropriate for in vivo delivery. Such considerations are well known in the art. IV. Hepatitis B Virus (HBV) infection and treatment [0408] As used herein, “Hepatitis B virus,” used interchangeably with the term “HBV” refers to the well-known non-cytopathic, liver-tropic DNA virus belonging to the Hepadnaviridae family. [0409] The HBV genome is partially double-stranded, circular DNA with overlapping reading frames. [0410] There are four transcripts (that may be referred to herein as “genes” or “open reading frames”) based on size, encoded by the HBV genome. These contain open reading frames called C, X, P, and S. The core protein is coded for by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. The DNA polymerase is encoded by gene P. Gene S is the gene that codes for the surface antigens (HBsAg). The HBsAg gene is one long open reading frame which contains three in frame “start” (ATG) codons resulting in polypeptides of three different sizes called large, middle, and small S antigens, pre-S1+pre-S2+S, pre-S2+S, or S. Surface antigens in addition to decorating the envelope of HBV, are also part of subviral particles, which are produced at large excess as compared to virion particles, and play a role in immune tolerance and in sequestering anti-HBsAg antibodies, thereby allowing for infectious particles to escape immune detection. The function of the non-structural protein coded for by gene X is not fully understood, but it plays a role in transcriptional transactivation and replication and is associated with the development of liver cancer. [0411] HBV is one of the few DNA viruses that utilize reverse transcriptase in the replication process which involves multiple stages including entry, uncoating, and transport of the virus genome to the nucleus. Initially, replication of the HBV genome involves the generation of an RNA intermediate that is then reverse transcribed to produce the DNA viral genome. [0412] Upon infection of a cell with HBV, the viral genomic relaxed circular DNA (rcDNA) is transported into the cell nucleus and converted into episomal covalently closed circular DNA (cccDNA), which serves as the transcription template for the viral mRNAs. After transcription and nuclear export, cytoplasmic viral pregenomic RNA (pgRNA) is assembled with HBV polymerase and capsid proteins to form the nucleocapsid, inside which polymerase-catalyzed reverse transcription yields minus-strand DNA, which is subsequently copied into plus-strand DNA to form the progeny rcDNA genome. The mature nucleocapsids are then either packaged with viral envelope proteins to egress as virion particles or shuttled to the nucleus to amplify the cccDNA reservoir through the intracellular cccDNA amplification pathway. cccDNA is an essential component of the HBV replication cycle and is responsible for the establishment of infection and viral persistence. [0413] HBV infection results in the production of two different particles: 1) the infectious HBV virus itself (or Dane particle) which includes a viral capsid assembled from the HBcAg and is covered by an envelope consisting of a lipid membrane with HBV surface antigens, and 2) subviral particles (or SVPs) which contain the small and medium forms of the hepatitis B surface antigen HBsAg which are non-infectious. For each viral particle produced, over 10,000 SVPs are released into the blood. As such, SVPs (and the HBsAg protein they carry) represent the overwhelming majority of viral protein in the blood. HBV infected cells also secrete a soluble proteolytic product of the pre-core protein called the HBV e-antigen (HBeAg). [0414] Eight genotypes of HBV, designated A to I, have been determined, and an additional genotype J has been proposed, each having a distinct geographical distribution. The virus is non-cytopathic, with virus-specific cellular immunity being the main determinant for the outcome of exposure to HBV− acute infection with resolution of liver diseases with 6 months, or chronic HBV infection that is frequently associated with progressive liver injury. [0415] The term “HBV” includes any of the genotypes of HBV (A to J). The complete coding sequence of the reference sequence of the HBV genome may be found in for example, GenBank Accession Nos. GI:21326584 and GI:3582357. Amino acid sequences for the C, X, P, and S proteins can be found, for example at NCBI Accession numbers YP_009173857.1 (C protein); YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P protein); and YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein). [0416] Additional examples of HBV mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM. The International Repository for Hepatitis B Virus Strain Data can be accessed at www.hpa- bioinformatics.org.uk/HepSEQ/main.php. [0417] The term “HBV,” as used herein, also refers to naturally occurring DNA sequence variations of the HBV genome, i.e., genotypes A-J and variants thereof. A study analyzing over 900 publications was performed and data were extracted from 213 records covering 125 countries (Genes 2018, 9(10), 495; doi.org/10.3390/genes9100495). Using previously published HBV prevalence, and population data, the number of infections with each HBV genotype per country was approximated and the genotype distribution among global chronic HBV infections. It was estimated that 96% of chronic HBV infections worldwide are caused by five of the nine genotypes confirmed at that time: genotype C is most common (26%), followed by genotype D (22%), E (18%), A (17%) and B (14%). Genotypes F to I together were estimated to cause less than 2% of global chronic HBV infections. [0418] The term “hepatitis D virus” or “HDV” as used herein is a circular, single stranded RNA virus that ranges from 1,672 (strain dFr45, Genbank accession number AX741144) to 1,697 nucleotides (dFr47, GenBank accession number AX741149). A unique open reading frame encodes the small and large hepatitis delta (sHD and IHD, respectively) antigens by way of an editing step in the hepatocyte nucleus. [0419] The genetic diversity of HDV is related to the geographic origin of the isolates and there are at least eight genotypes that are referred to as HDV-1 through HDV-8. Apart from HDV-1, which is ubiquitous, HDV-2 (previously labeled HDV-IIa) is found in Japan, Taiwan, and Yakoutia, Russia; HDV-4 (previously labeled HDV-IIb) in Taiwan and Japan; HDV-3 which causes epidemics of severe and fulminant hepatitis in the Amazonian region (9); and HDV-5, HDV-6, HDV- 7, and HDV-8 in Africa (LeGal et al., Emerg. Infect. Dis. 12:1447-1450, 2006). [0420] As used herein, the term "Hepatitis D virus-associated disease" or "HDV- associated disease," is a disease or disorder that is caused by, or associated with HDV infection or replication. The term "HDV-associated disease" includes a disease, disorder or condition that would benefit from reduction in HDV gene expression or replication. Non- limiting examples of HDV-associated diseases include, for example, hepatitis B virus infection, acute hepatits B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; hepatocellular carcinoma. [0421] The “WHO Guidelines for prevention, care and treatment of persons with chronic Hepatitis B Infection, 2015” is used herein as a source for information and definitions related to hepatitis B virus, hepatitis B infection, and compositions and methods for treatment of hepatitis B infection. Further information related to the same are provided therein. [0422] “Acute HBV infection” is used herein as new-onset hepatitis B infection that may or may not be icteric or symptomatic. Diagnosis is based on detection of hepatitis B surface antigen (HBsAg) and IgM antibodies to hepatitis B core antigen (anti-HBc). Recovery is accompanied by clearance of HBsAg with seroconversion to anti-HBs (antibodies to hepatitis B surface antigen), usually within 3 months. [0423] “Chronic HBV infection” or “chronic hepatitis B” is used herein as persistence of hepatitis B surface antigen (HBsAg) for six months or more after acute infection with HBV. [0424] “Immune-tolerant phase” as used herein is the high replicative phase of infection seen in the early stage of CHB among people infected at birth or in early childhood. [0425] “Immune-active phase” as used herein is the phase of hepatitis B e antigen (HBeAg)-positive disease characterized by fluctuating aminotransferases and high HBV DNA concentrations. May result in seroconversion from HBeAg to anti-HBe (antibody to hepatitis B e antigen). [0426] “Inactive phase” or “immune control phase” as used herein is the low replicative phase of chronic hepatitis B characterized by HBeAg negativity, anti-HBe positivity, normal alanine aminotransferase (ALT) and HBV DNA concentration below 2000 IU/mL. [0427] “HBeAg seroconversion” as used herein is the loss of HBeAg and seroconversion to anti-HBe-positive. [0428] “HBeAg-negative chronic hepatitis B (immune escape phase)” as used herein is HBV infection in a subject that is HBeAg-negative, but anti-HBe-positive disease with variable levels of HBV replication and liver injury. [0429] “HBsAg seroconversion” as used herein is understood as the loss of HBsAg and seroconversion to anti-HBs positive. [0430] “HBeAg reversion” as used herein is understood as the reappearance of HBeAg in a person who was previously HBeAg negative, and usually associated with increased HBV replication. [0431] “Cirrhosis” as used herein is an advanced stage of liver disease characterized by extensive hepatic fibrosis, nodularity of the liver, alteration of liver architecture and disrupted hepatic circulation. [0432] “Decompensated cirrhosis” is understood as a clinical complications of cirrhosis become manifest, including jaundice, ascites, spontaneous bacterial peritonitis, oesophageal varices and bleeding, hepatic encephalopathy, sepsis and renal failure [0433] “Hepatitis B surface antigen (HBsAg)” HBV envelope protein and excess coat particles detectable in the blood in acute and chronic hepatitis B infection. [0434] “Hepatitis B core antigen (HBcAg)” HBV core protein. The core protein is coated with HBsAg and therefore not found free in serum. [0435] “Hepatitis B e antigen (HBeAg)” Viral protein found in the high replicative phase of hepatitis B. HBeAg is usually a marker of high levels of replication with wild type virus, but is not essential for viral replication. [0436] “Hepatitis B surface antibody (anti-HBs)” as used herein is understood as an antibody that binds specifically to HBsAg. Anti-HBs develop in response to HBV vaccination and during recovery from acute hepatitis B, denoting past infection and immunity. [0437] “Hepatitis B e antigen antibody” or “Anti-HBe”as used herein is understood as an antibody that binds specifically to HBeAg Detected in persons with lower levels of HBV replication but also in HBeAg-negative disease (i.e. HBV that does not express HBeAg). [0438] “Hepatitis B core antibody (anti-HBc)” as used herein is understood as an antibody to hepatitis B core (capsid) protein. Anti-HBc antibodies are not neutralizing antibodies and are detected in both acute and chronic infection. [0439] “IgM anti-HBc” is a subclass of anti-HBc that is detected in acute hepatitis B, but can be detected by sensitive assays in active chronic HBV. [0440] “IgG anti-HBc” is a subclass of anti-HBc that is detected in past or current infection. [0441] “Occult HBV infection” is a persistent infection present in persons who have cleared hepatitis B surface antigen, i.e. they are HBsAg negative but HBV DNA positive, although at very low levels (invariably <200 IU/mL). Most are also anti-HBc positive. [0442] “Treatment failure” as used herein includes both primary or secondary treatment failure. In settings where HBV DNA testing is available: Primary antiviral treatment failure may be defined as failure of an antiviral drug to reduce HBV DNA levels by ^1 x log10 IU/mL within 3 months of initiating therapy. Secondary antiviral treatment failure may be defined as a rebound of HBV DNA levels of ≥1 x log10 IU/mL from the nadir in persons with an initial antiviral treatment effect (≥1 x log10 IU/mL decrease in serum HBV DNA). [0443] In settings where HBV DNA testing is not available: Treatment failure and drug resistance may be suspected based on features including: receiving antiviral drugs with a low barrier to resistance together with documented or suspected poor adherence, laboratory measures such as an increase in serum aminotransferases, or evidence of progressive liver disease. However, elevation in ALT level tends to occur late and is a relatively poor predictive marker of resistance. Confirmation of antiviral drug failure can be established, for example, by sequencing the HBV DNA polymerase and identifying specific genetic markers of antiviral drug resistance. [0444] “Persistently abnormal or normal ALT level” as used herein is an assessment of ALT levels over time. ALT levels fluctuate in persons with chronic hepatitis B and require longitudinal monitoring to determine the trend. Upper limits for normal ALT have been defined as below 30 U/L for men and 19 U/L for women, although local laboratory normal ranges should be applied. Persistently abnormal or normal may be defined as three ALT determinations above or below the upper limit of normal, respectively, made at unspecified intervals during a 6–12-month period or predefined intervals during a 12-month period. [0445] As used herein, the term “nucelot(s)ide analog” or “reverse transcriptase inhibitor” is an inhibitor of DNA replication that is structurally similar to a nucleotide or nucleoside and specifically inhibits replication of the HBV cccDNA and does not significantly inhibit the replication of the host (e.g., human) DNA. Such inhibitors include Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (TAF), Lamivudine, Adefovir dipivoxil, Entecavir (ETV), Telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-Acetyl-Cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380/LB-80380), and tenofvir-exaliades (TLX/CMX157). In certain embodiments, the nucelot(s)ide analog is Entecavir (ETV). Nucleot(s)ide analogs are commercially available from a number of sources and are used in the methods provided herein according to their label indication (e.g., typically orally administered at a specific dose) or as determined by a skilled practitioner in the treatment of HBV. [0446] As used herein, a “therapeutic HBV vaccine,” and the like, can be a peptide vaccine, a DNA vaccine including a vector-based vaccine, or a cell-based vaccine that induces an immune response, preferably an effector T cell induced response, against one or more HBV proteins. Preferably the vaccine is a multi-epitope vaccine that is cross-reactive with multiple HBV serotypes, preferably all HBV serotypes. A number of therapeutic HBV vaccines are known in the art and are at various stages of pre-clinical and clinical development. Protein based vaccines include hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) vaccines (e.g., Li et al., 2015, Vaccine.33:4247-4254, incorporated herein by reference). Exemplary DNA vaccines include HB-110 (Genexine, Kim et al., 2008. Exp Mol Med.40: 669-676), pDPSC18 (PowderMed), INO-1800 (Inovio Pharmaceuticals), HBO2 VAC-AND (ANRS), and CVI-HBV-002 (CHA Vaccine Institute Co., Ltd.). Exemplary protein based vaccines include Theravax/DV-601 (Dynavax Technologies Corp.), EPA-44 (Chongqing Jiachen Biotechnology Ltd.), and ABX 203 (ABIVAX S.A.). Exemplary cell based vaccines include HPDCs-T immune therapy (Sun Yat-Sen University). Combination vaccines and products are also known and include HepTcell™ (FP-02.2 vaccine (peptide)+IC310 Adjuvant (a combination peptide-oligonucleotide adjuvant), (see U.S. Patent Publication Nos.2013/0330382, 2012/0276138, and 2015/0216967, the entire contents of each of which is incorporated herein by reference)); GS-4774 (Gilead, a fusion protein S. core X vaccine+Tarmogen T cell immune stimulator), pSG2.HBs/MVA.HBs (protein prime/viral vector boost, Oxxon Therapeutics), and a protein-prime/modified vaccinia virus Ankara vector-boost (HBsAg and HBsAg protein+HBcAg and HBsAg in MVA expression vector, Backes et al., 2016, Vaccine.34:923-32, and WO2017121791, both of which are incorporated herein by reference). [0447] As used herein, the term “adjuvant” is understood to be an agent that promotes (e.g., enhances, accelerates, or prolongs) an immune response to an antigen with which it is administered to elicit long-term protective immunity. No substantial immune response is directed at the adjuvant itself. Adjuvants include, but are not limited to, pathogen components, particulate adjuvants, and combination adjuvants (see, e.g., www.niaid.nih.gov/research/vaccine-adjuvants-types). Pathogen components (e.g., monophosphoryl lipid A (MPL), poly(I:C), polyICLC adjuvant, CpG DNA, c-di-AMP, c-di- GMP, c-di-CMP; short, blunt-ended 5^-triphosphate dsRNA (3pRNA) RIG-1 ligand, and emulsions such as poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP)) can help trigger early non-specific, or innate, immune responses to vaccines by targeting various receptors inside or on the surface of innate immune cells. The innate immune system influences adaptive immune responses, which provide long-lasting protection against the pathogen that the vaccine targets. Particulate adjuvants (e.g., alum, virosomes, cytokines, e.g., IL-12) form very small particles that can stimulate the immune system and also may enhance delivery of antigen to immune cells. Combination adjuvants (e.g., AS02, AS03, and AS04 (all GSK); MF59 (Novartis); ISCOMATRIX® (CSL Limited); and IC31® (Altimmune) elicit multiple protective immune responses. Adjuvants that have a modest effect when used alone may induce a more potent immune response when used together. In certain embodiments, adjuvants promote a humoral as well as a cellular immune response. For this, a balanced Th1/Th2 helper T cell response is desired to support neutralizing antibody responses as well as effector cytotoxic T cell responses. Such adjuvants include, for example, polyI:C adjuvant, a polyICLC adjuvant, a CpG adjuvant, a STING agonist (a c-di- AMP adjuvant, a c-di-GMP adjuvant, or a c-di-CMP adjuvant), an ISCOMATRIX® adjuvant, a PCEP adjuvant, and a Rig-I-ligand adjuvant. In certain embodiments, the adjuvant is a polyI:C adjuvant, a CpG adjuvant, a STING agonist, or a PCEP adjuvant. In certain embodiments, the adjuvant is not alum. [0448] As used herein, an “immune stimulator” is an agent that stimulates an immune response that may or may not be administered independently of an antigen. Immune stimulators include, but are not limited to, pegylated interferon alfa 2a (PEG-IFN-alpha-2a), interferon alfa-2b, PEG-IFN-alpha-2b, interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor (e.g., Myrcludex), an oligonucleotide that inhibits the secretion or release of HBsAg (e.g., REP 9AC), a capsid inhibitor (e.g., Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g., IHVR-25). In certain embodiments, an immune stimulator can include a viral capsid, optionally an empty viral capsid, e.g., MVA capsid. [0449] Immune stimulators can also include immune checkpoint regulators. Immune checkpoint regulators can be stimulatory or inhibitory. As used herein, immune checkpoint regulators potentiate an immune response Immune checkpoint regulators include, but are not limited to, CTLA-4 inhibitors, such as ipilimumab, PD-1 inhibitors, such as Nivolumab, Pembrolizumab, and the BGB-A317 antibody. PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab, in addition to an affimer biotherapeutic. [0450] In certain embodiments, an HBV-associated disease is chronic hepatitis B (CHB). In certain embodiments, subjects have been infected with HBV for at least five years. In certain embodiments, subjects have been infected with HBV for at least ten years. In certain embodiments, subjects became infected with HBV at birth. In certain embodiments, an HBV-associated disease is HDV. [0451] Subjects having chronic hepatitis B disease are immune tolerant, have an active chronic infection accompanied by necroinflammatory liver disease, have increased hepatocyte turn-over in the absence of detectable necroinflammation, or have an inactive chronic infection without any evidence of active disease, and they are also asymptomatic. Subjects having chronic hepatitis B disease are HBsAg positive and have either high viremia (>10e4 HBV-DNA copies/ml blood) or low viremia (<10e3 HBV-DNA copies/ml blood). Patients with chronic active hepatitis, especially during the high replicative state, may have symptoms similar to those of acute hepatitis. The persistence of HBV infection in CHB subjects is the result of ccc HBV DNA persistence. In certain embodiments, a subject having CHB is HBeAg positive. In other embodiments, a subject having CHB is HBeAg negative. [0452] The term “lower” in the context of the level of HBV gene expression or HBV replication in a subject, or a disease marker or symptom, refers to a statistically significant decrease in such level. The decrease can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In monitoring of HBV infection, a log 10 scale is typically used to describe the level of antigenemia (e.g., HBsAg level in serum) or viremia (HBV DNA level in serum). It is understood that a 1 log 10 decrease is a 90% decrease (10% remaining), a 2 log 10 decrease is a 99% decrease (1% remaining), etc. In certain embodiments, a disease marker is lowered to below the level of detection. [0453] In certain embodiments, the expression of a disease marker is normalized, i.e., decreased to a level accepted as within the range of normal for an individual without such disorder, e.g., the level of a disease marker, such as, ALT or AST, is decreased to a level, preferably persistently decreased to a level, accepted as within the range of normal for an individual without such disorder. When the disease associated level is elevated from the normal level, the change is calculated from the upper level of normal (ULN). When the disease associated level is decreased from the normal level, the change is calculated from the lower level of normal (LLN). The lowering is the percent difference in the change between the subject value and the normal value. For example, a normal AST level can be reported as 10 to 40 units per liter. If, prior to treatment, a subject has an AST level of 200 units per liter (i.e., 5 times the ULN, 160 units per liter above the upper level of normal) and, after treatment, the subject has an AST level of 120 units per liter (i.e., 3 times the ULN, 80 units per liter above the upper level of normal), the elevated AST would be lowered towards normal by 50% (80/160). It is understood that the level of AST is variable, particularly in individuals having HBV. Persistently abnormal or normal may be defined as three AST determinations above or below the upper limit of normal, respectively, made at unspecified intervals during a 6–12-month period or predefined intervals during a 12-month period. [0454] The phrase “inhibiting expression of an HBV gene” is intended to refer to knockdown of any HBV transcript (e.g., 3.5 kb, 2.4 kb, 2.1 kb, or 0.7 kb transcript) encoding one or more HBV viral proteins (such as, e.g., preS1/2-S, preS, S, P, X, preC, and C), as well as variants or mutants of an HBV gene. [0455] “Inhibiting expression of an HBV gene” includes any significant level of inhibition of an HBV gene or transcript, e.g., at least partial suppression of the expression of an HBV gene S, P, X, or C, or any combination thereof, e.g., S, P, and C. The expression of the HBV gene may be assessed based on the level, or the change in the level, of any variable associated with HBV gene expression, e.g., an HBV mRNA level, an HBV protein level, or an HBV cccDNA level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject, e.g., levels may be monitored in serum. [0456] In some embodients, only one dose of the guide RNA or composition provided herein is adminietered to a subject with HBV infection to treat HBV infection. In some embodiments, more than one dose (e.g., 2, 3, 4, or 5 doses) of the guide RNA or composition provided herein are adminietered, and each dose may be separated by 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, or 24 weeks. For example, in some embodients, two doses of the guide RNA or composition provided herein is adminietered to a subject with HBV infection, and the second dose is administered after about 1 month, 3 month, or 6 month after the first dose. [0457] In some embodiments, the methods provided herein comprises (1) administering a first dose of the HBV-targeting guide RNA provided herein and an RNA- guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or a composition provided herein to a subject with HBV infection, (2) determining a clinical marker associated with HBV infection (e.g., serum HBV DNA or liver HBV ccc DNA, serum or liver HBV antigen, e.g., HBsAg or HBeAg), and (3) administering a second dose of the agent if the clinical marker indicates the presence of HBV infection (e.g., if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml). In some embodiments, the clinical marker associated with HBV infection is measured after a period of time after the first dose (e.g., 1 month, 3 months, or 6 months after the first dose). In some embodiment, the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose. In some embodiments, step (2) and step (3) can be repeated multiple rounds until the clinical marker indicates the absence or low HBV infection, for example e.g., if the HBsAg protein level or the HBcrAg protein level is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml). [0458] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained and tolerances accepted within the art. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. V. EXAMPLES Example 1. Materials and Methods Example 1.1. Next-generation sequencing (“NGS”) and analysis for on-target cleavage efficiency [0459] Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Cat. QE09050) according to the manufacturer's protocol. To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed around the target site within the gene of interest and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field. [0460] Additional PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to either the human reference genome (e.g., hg38) or HBV genotype D sequence contained in HepAD38 after eliminating those having low quality scores. Reads that overlapped the target region of interest were re-aligned to the local genome sequence to improve the alignment. Then the number of wild type reads versus the number of reads which contain C-to-T mutations, C-to-A/G mutations or indels was calculated. Insertions and deletions were scored in a 20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the 20 bp scoring region divided by the total number of sequencing reads, including wild type. C-to-T mutations or C-to-A/G mutations were scored in a 40 bp region including 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence. The C-to-T editing percentage is defined as the total number of sequencing reads with either one or more C-to-T mutations within the 40 bp region divided by the total number of sequencing reads, including wild type. The percentage of C-to-A/G mutations are calculated similarly. [0461] An additional computational analysis was conducted on the NGS data to predict the potential phenotypic outcome of C-to-T editing (i.e., percent stop codon gained or percent start codon loss). Editing consequences were evaluated on the read level for each isoform. Example 1.2. Preparation of lipid nanoparticles [0462] In general, the lipid nanoparticle (LNP) components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPs used contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), also called herein Lipid A, cholesterol, distearoylphosphatidylcholine (DSPC), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA) in a molar ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The LNPs used comprise a single RNA species such as Cas9 mRNA or a sgRNA. LNP are similarly prepared with a mixture of Cas9 mRNA and a guide RNA. [0463] The LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. Then, a fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840 FIG.2). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting mixture was then filtered using a 0.2 ^m sterile filter. The final LNPs were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size. The final LNP was stored at 4°C or -80°C until further use. Example 1.3. In vitro transcription (“IVT”) of mRNA [0464] Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The linearized plasmid DNA containing a T7 promoter, and a sequence for transcription was linearized by restriction endonuclease digestion followed by heat inactivation of the reaction mixture and purified from enzyme and buffer salts. Messenger RNA was synthesized and purified using standard techniques known in the art. [0465] Streptococcus pyogenes (“Spy”) Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs:706 or 712 (see sequences in Table 40). When SEQ ID NOs: 706, 712, or other SEQ ID NOs encoding open reading frames, are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above). Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NO: 701 in Table 40. [0466] Neisseria meningitidis (Nme) Cas 9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 710 or 715 (see sequences in Table 40). When SEQ ID NO: 710, 715, or other SEQ ID NOs encoding open reading frames, are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above). Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NOs: 703 and 704 in Table 40. [0467] Uracil glycosylase inhibitor (UGI) was generated from plasmid DNA encoding an open reading frame according to SEQ ID NO: 708. When SEQ ID NO: 708, or other SEQ ID NOs encoding open reading frames, are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above). Messenger RNAs used in the Examples include a 5’ cap and a 3’ poly-A tail, e.g., up to 100 nts, and are identified by the SEQ ID NO: 702 in Table 40. [0468] Guide RNAs are chemically synthesized by methods known in the art. Example 1.4. HepAD38 Preparation for Transfection and Experiment Timeline [0469] HepAD38 (Ladner et al.1997 Antimicrobial Agents and Chemotherapy 41(8): 1715-1720), a human liver cell line licensed from Fox Chase Cancer Center, contains an integrated, tetracycline-responsive, CMV-IE-promoter-driven copy of HBV Genotype D, ayw strain. Complete medium for the HepAD38 cell line was composed of DMEM/F121:1 media (Corning 10-092-CM) containing 10% Tet-safe fetal bovine serum (Gibco, A4736401), 1X non-essential amino acids (Corning, 25-025-C1), 1 mM Sodium Pyruvate (Corning 25-000-Cl), and 1X Penicillin-Streptomycin (Gibco 15070-063). Complete media was supplemented with 1 mg/mL Doxycycline (Sigma-Aldrich, D9891) to suppress inducible surface antigen production and 250 µg/mL G418 Sulfate selection marker (Corning, 30-234- CR) to eliminate cells that did not carry the integrated copy of HBV. HepAD38 cells were thawed and passaged in complete media with supplements in a 10 cm collagen coated dish. Prior to plating cells for transfection, complete media without supplements was prepared and cells were plated in 96-well collagen coated plates at a density of 20,000 cells/well. Supplements were removed prior to transfection to induce surface antigen production and remove elements that may interfere with the transfection or cell health. [0470] A typical experimental timeline for HepAD38 experiments was as follows. Cells were plated as described above on Day 0. Transfection was performed 24 hours later on Day 1. On Day 4, upon completion of editing, transfection media was removed from the plate and replaced with fresh complete media without supplements. The cells were maintained for another 7 days to allow for surface antigen accumulation in cell culture media. On Day 11, cell culture media was collected and stored at -80°C for downstream assays. Cells were lysed in 50 µL/well QuickExtract™ DNA Extraction Solution (Lucigen, QE09050) and stored at - 80°C for downstream assays. If a downstream immunofluorescence assay was performed, a replicate set of plates was prepared, and the cells were subjected to fixing and staining as described in Example 1.7. Example 1.5. Preparation of Viral Inoculum^^ [0471] HepAD38 cells carry the Hepatitis B virus integrated into the genome. To harvest viral particles, 3 million HepAD38 cells were plated in a collagen-coated 10 cm dish in complete cell media described in Example 1.4 (without supplements). After seven days, media was harvested and replaced with fresh media (cells are not split). After seven days, media was harvested again. Harvested media was stored at -80^.^^ [0472] To precipitate viral particles, harvested media was centrifuged for 10 min at 1,500 x g to pellet cell debris. The supernatant was moved to a new tube and one volume of PEG-it™ Virus Precipitation Solution (System Biosciences, LV810A-1) was added to 4 volumes of supernatant. The tube was inverted several times and stored at 4^ overnight. The following day, the mixture was centrifuged at 1,500 x g for 30 min at 4^ to pellet the viral precipitate. The supernatant was aspirated and the pellet was resuspended in 1/10 of the original volume in complete media. The resulting viral inoculum was aliquoted and stored at -80^. Purified virus particle samples were titered (viral particles/mL) by isolating viral genomes with DNA minprep kits (Zymo Research D4003) and quantified using qPCR against a plasmid standard encoding the same HBV DNA sequence with primer sequences Fwd: 5’- GTTGCCCGTTTGTCCTCTAATTC-3’ (SEQ ID NO: 699), Rev: 5’- GGAGGGATACATAGAGGTTCCTTGA-3’ (SEQ ID NO: 700). Example 1.6. HBsAg MSD ELISA Protocol [0473] This MSD assay was used to determine HBsAg concentrations in either cell culture media samples or in mouse serum samples. [0474] Capture antibody (Monoclonal Anti-HBV antigen HBsAg antibody produced in mouse, Sigma, SAB4700767) and detector antibody (Anti-Hepatitis B Virus Surface Antigen antibody produced in mouse, Abcam, ab8256) were buffer exchanged to PBS using Amicon Centrifugal Unit (Sigma, UFC5050) prior to use. [0475] Biotinylation: One tube containing the biotinylation labeling reagent (Thermo Fisher, A39259) was thawed at 4^. A 2 nmol/µL capture solution was prepared by mixing 6.7 µl of biotin solution into 100 µl 1 mg/ml buffer exchanged capture antibody. Capture solution was incubated at room temperature for 2 hours in the dark, then was kept at 4^ overnight. [0476] Amicon centrifugal unit was used to remove residual biotin. [0477] Sulfo-tag conjugation: A 3 nmol/µL sulfo-tag (Meso Scale Discovery, R91AO-1) solution was prepared in cold dH2O.13.4 µL of the sulfo-tag solution was added into 100 µL of 1 mg/mL buffer exchanged detector antibody. Detector solution was incubated at room temperature in the dark for 2 hours, then was kept at 4^ overnight. Amicon centrifugal unit was used to remove residual sulfo-tag. [0478] Blocker B Buffer: 1% and 0.5% Blocker B Buffer (Sigma, RPN2125V) solutions were prepared in PBST. A 2 µg/mL solution of capture antibody was prepared in 0.5% Blocker B buffer.25 µL/well of the capture solution was used to coat the plate (Meso Scale Discovery, L15SA-1). The sealed plate was shaken at 700 rpm at room temperature for 1 hour. [0479] Capture solution was removed and 150 µL of 1% Blocker B buffer was added per well. Sealed plate was shaken at 700 rpm at room temperature for 30 min. Plate was washed 3x with 200 µL/well PBST. [0480] In Vitro Standard and Sample Preparation: Standard curve was prepared in 0.5% Blocker B buffer using (CellBioLabs- Recombinant HBsAg Standard, 50043C). The standard ranged from 500 ng/mL to 0 ng/mL using a 5-fold serial dilution scheme. In vitro samples were diluted 1:5 in 0.5% Blocker B buffer.50 µL/well of either standard or sample was added to the plate (standards were run in duplicate). The sealed plate was shaken at 700 rpm at room temperature for 1 hour. Plate was washed 3x with 200 µL/well PBST. [0481] In Vivo Standard and Sample Preparation: 20 µL of mouse serum (BioIVT, MSE01SRMPNN) was added to 980 µL of 0.5% Blocker B buffer. Standard was prepared as described in In Vitro Standard Preparation using the mouse serum buffer. In Vivo samples were diluted 1:50 in 0.5% Blocker B buffer.50 µL/well of either standard or sample was added to the plate (standards were run in duplicate). The sealed plate was shaken at 700 rpm at room temperature for 1 hour. Plate was washed 3x with 200 µL/well PBST. [0482] Detector: A 2 µg/mL solution of detector antibody was prepared in 0.5% Blocker B Buffer.25 µL of detector solution was added per well. The sealed plate was shaken at 700 rpm at room temperature for 1 hour. Plate was washed 3x with 200 µL/well PBST. [0483] Reading: 150 µL/well of 1x MSD Reading buffer (Meso Scale Discovery, R92TC-1) was added to the plate and read on QuickPlex SQ120 system. Example 1.7. Immunofluorescence Assay for Intracellular HBsAg Detection Via Microscopy [0484] HepAD38 cells were prepared as in Example 1.4, fixed and stained for intracellular HBsAg detection via microscopy post transfection. Levels of HBsAg in cells treated with a S-Antigen knockdown guide were evaluated against untreated cells by high content microscopy (CX7 LZR, ThermoFisher). [0485] Cells were rinsed with PBS and then fixed using a 4% paraformaldehyde solution in PBS and incubated at room temperature for fifteen minutes. Cells were washed twice with PBS. to permeabilize the cells a 0.5% TritonX100 solution in PBS was prepared and incubated at room temperature on the cells for twenty minutes. Cells were washed twice with 100uL PBS. Blocking was performed using DAKO Blocking Buffer (DAKO, X090930- 2) with slow rocking at room temperature for thirty minutes. The primary antibody, a Mouse anti-HBsAg antibody (Sigma, SAB4700767), was diluted 1:400 in DAKO Antibody Dilution Buffer (DAKO, K800621-2). The blocking buffer was removed from the plate and 50 µL of the primary antibody solution was added and allowed to incubate with rocking overnight at 4^. The following day, the plate was washed twice with 0.05% Tween20 in PBS, then washed twice with PBS. A secondary antibody solution was prepared by diluting a Goat anti- Mouse Alexa Fluor™ Plus 488 antibody 1:500 (ThermoFisher Scientific, A32723) and Hoechst stain 1:10,000 (ThermoFisher Scientific, 33342) in Antibody Dilution Buffer. Secondary antibody/Hoechst solution was added to the plate and incubated for about two hours at room temperature with slow shaking. The plate was washed twice with 0.05% Tween20 in PBS and allowed to incubate with the second Tween20 wash for fifteen minutes at room temperature. PBS was added to the plate and imaged and analyzed using a CX7 high- content microscope with SpotDetector Bioassay for intracellular HBsAg detection. Example 2. Guide screen for HBV S-antigen knockout in HepAD38 cells using Spy and Nme2 base editors and cleavases [0486] To evaluate S. pyogenes (SpyCas9) C-to-T editing, Spy-BC22n, an mRNA encoding Spy Cas9 base editor (open reading frame SEQ ID NO: 706), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), and each gRNA from a set of Spy gRNAs targeting the integrated HBV genome (Table 17) were transfected together. Similarly, to evaluate N. meningitidis (Nme2Cas9) C-to-T editing, Nme2-BC22n, an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 715), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), and each gRNA from a set of Nme2 gRNAs targeting the integrated HBV genome (Table 17) were transfected. Additionally, C-to-T editing with Spy and Nme2 was compared to indel formation with their respective cleavases, either an mRNA encoding Spy Cas9 (open reading frame SEQ ID NO: 712) or an mRNA encoding Nme2 Cas9 (open reading frame SEQ ID NO: 710). Spy sgRNAs included chemical modifications shown in Table 12. Nme sgRNAs included the chemical modifications shown in Table 14. Table 17: List of guide RNAs tested
Figure imgf000175_0001
Figure imgf000176_0001
Example 2.1. Cell preparation and Transfection [0487] HepAD38 cells were thawed, passaged and transfected as described in Example 1.4 with lipoplexes formed with UGI, a gRNA targeting the HBV surface antigen in the integrated HBV genome of HepAD38, and the appropriate nuclease Spy-BC22n or Nme2-BC22n for the guide RNA. Cleavase lipoplexes also contained cleavase mRNA, UGI mRNA, and guide at the same RNA ratios. [0488] Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Lipofection reagent was combined with a cargo by mixing each lipid-formulated RNA species at a 1:1:1 weight ratio: effector mRNA (base editor or cleavase): UGI mRNA: gRNA. The materials were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The resulting bulk-mixed lipoplex material (lipid kits) was pre-incubated with 10% FBS (Corning, 35-101-CV) in complete media (plus Doxycycline) at 37^ for 10 minutes before addition to HepAD38 cells. [0489] For the screen, each well received 100 ng of each individual RNA cargo in 100 µL of complete media. Example 2.2. Evaluation of C-to-T editing outcomes by next generation sequencing (NGS) [0490] Ninety-six hours post transfection, cells were subjected to lysis, PCR amplification of the target site in the integrated HBV genome and subsequent NGS analysis, as described in Example 1.1. All experiments were performed in biological duplicates. Table 18 and Figure 1 show the NGS sequencing and predicted phenotypic outcomes for all guides targeting the HBV S-Antigen with either Spy-BC22n or Spy-cleavase. Table 19 and Figure 2 show the NGS sequencing and predicted phenotypic outcomes for all guides targeting the HBV S-Antigen with Nme2-BC22n and Nme2-cleavase. Table 18- Genome editing and predicted phenotypic outcomes for guides targeting HBV S-Antigen with Spy-BC22n and Spy-cleavase
Figure imgf000177_0001
Table 19- Genome editing and predicted phenotypic outcomes for guides targeting HBV S-Antigen with Nme2-BC22n and Nme2-cleavase
Figure imgf000177_0002
Figure imgf000178_0001
Example 3. Dose-dependent editing of HBV guides with base editors in HepAD38 cells [0491] HepAD38 cells were transfected with gRNA dose-response curve to determine the relative potency, maximum C-to-T editing and maximum predicted knockout level of gRNA targeting the integrated S-antigen locus of HepAD38. Example 3.1: Cell preparation and transfection [0492] HepAD38 cells were thawed, passaged and transfected as described in Example 1.4 with a combination of an mRNA encoding Spy Cas9 base editor (SEQ ID NO:706), an mRNA encoding UGI (SEQ ID NO: 708) and a Spy gRNA targeting the integrated S-antigen locus of HepAD38, or a combination of an mRNA encoding Nme2 Cas9 base editor (SEQ ID NO: 715), an mRNA encoding UGI (SEQ ID NO: 708), and a gRNA targeting the integrated S-antigen locus of HepAD38. [0493] Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Lipofection reagent was combined by mixing separately each RNA species: base editor mRNA (Spy- or Nme2-BC22n), UGI mRNA, or gRNA. The materials were combined at a lipid amine to RNA phosphate (N:P) molar ratio of about 6. The resulting bulk-mixed lipoplex material was pre-incubated with 10% FBS (Corning, 35-101-CV) in complete media (plus Doxycycline) at 37^ for 10 min before addition to HepAD38 cells. [0494] For the guide titration, each well received either Spy- or Nme2-BC22n, and UGI at a final concentration of 100 ng/well each. Two guide RNA dose response curves were performed with concentrations ranging from either 100 nM to 0 nM or 10 nM to 0 nM, titrated using a 2-fold dilution scheme. The total volume of all three components delivered was 100 µL/well. Example 3.2: Evaluation of C-to-T editing outcomes by next generation sequencing (NGS) [0495] Seventy-two hours post transfection, cells were subjected to lysis, PCR amplification of the HBV S-Antigen locus, and subsequent NGS analysis, as described in Example 1.1. All experiments were performed in biological replicates (n=2). Two dose response curves are shown, with guide titration ranging from either 100 nM to 0 nM or 10 nM to 0 nM. [0496] Figures 3A and 3B and Tables 20A and 20B show editing starting at a dose of 100 nM guide using Spy-BC22n or Nme2-BC22n base editor at a constant dose. [0497] Figures 4A and 4B and Tables 21A and 21B show editing starting at a dose of 10 nM for both Spy-BC22n and Nme2-BC22n HBV S-antigen guides. Table 20A- Percent C-to-T Editing for Spy-BC22n 100 nM Dose Response Curve
Figure imgf000179_0001
Table 20B- Percent C-to-T Editing for Nme2-BC22n 100 nM Dose Response Curve
Figure imgf000180_0001
Table 21A- Percent C-to-T Editing for Spy-BC22n 10 nM Dose Response Curve
Figure imgf000180_0002
Table 21B- Percent C-to-T Editing for Nme2-BC22n 10 nM Dose Response Curve
Figure imgf000180_0003
Figure imgf000181_0001
Example 4- Screening to identify gRNA that reduce HBsAg and total HBV DNA in HepAD38 using Nme2 and Spy base editor [0498] The following methods apply to two guide screens, one using Nme2 base editor (Nme2-BC22n) and the other using Spy base editor (Spy-BC22n). In each screen, gRNA targeting multiple regions of the HBV genome were evaluated for NGS sequencing outcomes as well as HBsAg and total HBV DNA knockdown. [0499] HepAD38 cells were thawed, passaged, plated, and transfected as described in Example 1.4. One background control well in each plate was plated with HepG2 (parental) cells at the same density as HepAD38 cells. The HepG2 cells served as a background subtraction control for downstream assays. Two sets of biological duplicates were prepared (four plates total). One set was prepared in clear collagen-coated 96-well plates for editing, qPCR and secreted HBsAg and another set in black-walled collagen-coated 96-well plates for the downstream immunofluorescence assay. [0500] Twenty-four hours later, cells were transfected simultaneously with separate lipoplexes formed individually with either an mRNA encoding Spy Cas9 base editor (open reading frame SEQ ID NO:706) or an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 715), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), and gRNA targeting the HBV genotype D sequence in HepAD38. Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Single-cargo lipofection reagent was prepared by mixing separately each RNA species: base editor mRNA (Spy- or Nme2-BC22n), UGI mRNA, or gRNA. Each well received 100 ng of base editor mRNA, 100 ng of UGI mRNA, and 25 nM of gRNA diluted in complete media. The media-lipofection reagent mixture was incubated at 37^ for 10 min before addition to the cells. The total volume of all three components was 100 µL per well. [0501] Media change, media collection, and cell lysis were performed as described in Example 1.4. The set of black-walled plates were prepared for cell fixing and staining for an intracellular HBsAg immunofluorescence assay as described in Example 1.7. An MSD assay evaluating HBsAg knockdown was performed on collected cell media as described in Example 1.6. Cell lysate was used to quantify intracellular total HBV DNA using a qPCR assay described in Example 1.5. Cell lysates were also prepared for NGS analysis as described in Example 1.1. [0502] Table 22A-22B and Table 23A-23B show the NGS outcomes, percent intracellular HBsAg knockdown, percent secreted HBsAg knockdown, and percent intracellular total HBV DNA knockdown for all guides tested using Spy base editor or Nme2 base editor, respectively. Figure 5A-5B and Figure 6A-6B show knockdown levels for both HBsAg and total intracellular HBV DNA for Nme2 base editor and Spy base editor screens, respectively. Guides with >90% HBsAg knockdown or >90% total HBV DNA knockdown were selected for follow-up experiments.
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Example 5 - In vitro evaluation of Nme2-BC22n /UGI + gRNA LNP potency for knockdown of secreted HBV HBsAg in a primary human hepatocyte HBV infection model [0503] Guide RNAs identified by screening in HepAD38 cells as described above were further analyzed for potency in primary human hepatocytes (PHH) using an in vitro infection model. Briefly, PHH were first infected with Hepatitis B inoculum, and after an incubation period, LNPs containing either GFP-mRNA or a combination of Nme2-BC22n mRNA + UGI mRNA + gRNA-targeting HBV were delivered to the cells. After LNP delivery the media was changed to fresh media and secreted HBsAg produced by either edited or control wells was measured to calculate relative knockdown of HBsAg as detailed below. The viral inoculum was prepared as in Example 1.5. 5.1. Infection of Primary Human Hepatocytes [0504] Primary Human Hepatocytes (PHH) were thawed and recovered in CHRMs media (Gibco, CM7000). Cells were resuspended in Primary Human Hepatocyte Plating Media consisting of William’s E media (Gibco, A1217601), hepatocyte plating supplements (Gibco, CM3000) and 5% fetal bovine serum (Gibco, A13450). PHH were plated in a collagen-coated 96-well plate at a density of 30,000 cells/well. After eighteen hours, plating media was removed and replaced with Primary Hepatocyte Maintenance media consisting of William’s E media and hepatocyte maintenance supplements (Gibco, CM4000), One day later, media was removed and replaced with maintenance media containing 2% DMSO for three hours. Media was removed and replaced with HBV inoculum-containing media consisting of maintenance media, 4% PEG-8000, 2% DMSO, and viral inoculum at an MOI of 50. Plates were then centrifuged at 1000xg for thirty minutes before being returned to the incubator. Twenty-four hours later, viral media was removed and replaced with media containing 2% DMSO and 2% FBS. The next day the same media change was repeated. 5.2. LNP Transfection [0505] Three days post HBV infection, infected PHH cells were transfected with single LNP formulations (formulated as described in Example 1.2) containing either an mRNA encoding GFP (open reading frame SEQ ID NO: 727; negative control) or a combination of gRNA, an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 721), and an mRNA encoding UGI (open reading frame SEQ ID NO: 724) at a 1:2:1 weight ratio. One Spy-BC22n formulation was compared to the Nme2 guides. In this condition the Spy-BC22n and gRNA were packaged in one formulation and UGI mRNA was formulated in a separate formulation. In all base editing conditions gRNA: BC22n: UGI were delivered at a 1:2:1 weight ratio. Wells received either 11, 33, or 100 ng of total RNA cargo. The LNPs were pre-incubated in maintenance media with 2% DMSO and 6% FBS for 10 minutes before being added to cells. The cells were incubated with the LNPs for four days at which time the media was removed and changed to fresh media with 2% DMSO and 2% FBS. 5.3. Measuring Secreted HBsAg and Relative Knockdown [0506] Four days after the last media change, day 17 of the experiment, secreted HBV S Antigen (HBsAg) was measured using a commercially available ELISA kit (LSBio, LS- F37979) according to the manufacturer’s protocols. Media from all conditions was initially diluted at 1:10. However, as a number of samples did not fall within the limit of detection of the assay, the assay was re-run with media at a 1:200 dilution. Bridging samples that were in the linear range from both experiments showed that there was an absolute drop in HBsAg detection on average of 6.1-fold between both ELISA runs. This factor was used to normalize values from both experiments. After normalization, absolute HBsAg levels (ng/mL) were calculated. The average HBsAg levels from HBV gRNA-treated groups was compared to the average HBsAg levels in the GFP-mRNA group to calculate relative percent HBsAg knockdown. Table 24 and Figure 7 show the relative percent HBsAg knockdown of all experimental groups. Table 24: Percent HBsAg knockdown relative to LNP-GFP mRNA condition on day 17
Figure imgf000219_0001
Figure imgf000220_0001
Example 6 - Evaluation of Nme2-BC22n and Spy-BC22n LNP in AAV8-HBV1.2 Mouse Model [0507] NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) male mice from Jackson Labs were dosed at 6-10 weeks of age with AAV8 and LNP subsequently as indicated, or vehicle (PBS + 0.001% Pluronic for AAV vehicle, TSS for LNP vehicle) via the lateral tail vein. AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein. LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 µl/gram body weight. At various times points post-treatment, blood was collected, and sera was isolated for certain analyses as described further below. 6.1. Human Hepatitis B Virus Surface Antigen (HBsAg) ELISA and Total DNA qPCR analysis [0508] For in vivo studies, blood was collected, and the sera was isolated as indicated. The total human Hepatitis B Virus Surface antigen (HBsAg) levels were determined using an HBsAg (Human) ELISA kit (LifeSpan Bio, Cat# LS-F37979) according to the manufacturer’s protocol. Serum HBsAg levels were quantitated off a standard curve using 4- parameter logistic fit and expressed as µg/mL of serum. For qPCR, serum was diluted 1:50 and DNA was isolated using the QE lysis protocol (Lucigen, Cat # QE09050). qPCR was run using Absolute qPCR SYBR Green Rox Mix (Thermo Scientific, Cat #AB-1163/A) with a primer set (Fwd-5’-GTTGCCCGTTTGTCCTCTAATTC-3’ (SEQ ID NO: 699), Rev- 5’- GGAGGGATACATAGAGGTTCCTTGA-3’ (SEQ ID NO: 700)) against a highly conserved area of the HBV genome not edited by the tested gRNA. A standard plasmid encoding the same HBV sequence was used to determine DNA copies/mL. [0509] Seven sgRNA (one control, two Nme2-BC22n, four Spy-BC22n) were assessed for editing via the knock-down of episomal expression of human Hepatitis B Virus Surface antigen (HBsAg) from AAV8-HBV1.2 (SignaGen Laboratories Cat# SL100862) administered as described herein at a dose of 1.2e11 vg/mouse, as measured by ELISA. Three weeks post administration of AAV8-HBV material, serum HBsAg expression was assessed for baseline levels via HBsAg ELISA. LNPs tested in this Example were prepared and delivered to mice as described herein one week post the assessment of HBsAg baselines. Two LNPs, one containing gRNA and either an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 729) or an mRNA encoding Spy Cas9 base editor (open reading frame SEQ ID NO: 706), and another containing an mRNA encoding UGI (open reading frame SEQ ID NO: 732), were pre-mixed and administered at a dose level of 0.5 mg/kg (with respect to the total RNA cargo content) per body weight of animal. The weight ratio of gRNA:BC22n:UGI was 1:2:0.3. Thereafter, serum HBsAg expression was determined by ELISA at one, two, and four weeks post sgRNA administration. Five weeks post LNP dose, the animals were euthanized; blood was collected to assess terminal HBsAg and HBV DNA levels in serum. Liver was collected for Next Generation Sequencing (NGS) and editing evaluation at HBV target sites. C-to-T %, C-to-A/G % and indel % was determined by NGS as described in Example 1.1. Phenotypic measurements and editing at the guide-specific target sites are shown in Table 25 and Table 26. HBsAg time course is shown in Figure 8. Log10 knockdown of total HBV DNA at week 5 is shown in Figure 9. Finally, levels of knockdown were highly correlated between HBsAg and Total DNA levels in serum at study endpoint as shown in Figure 10. Table 25- Serum HBsAg time course, serum HBV DNA at endpoint and editing profile at endpoint.
Figure imgf000221_0001
Figure imgf000222_0001
Table 26: NGS C-to-T %, C-to-A/G % and indel %
Figure imgf000222_0002
Figure imgf000223_0001
Example 6.2. Evaluation of Nme2-BC22n LNP in a multidose experiment [0510] Four individual sgRNA (G027224, G030107, G030115, G030120) and one multiplex sgRNA pair (G030107/G030120) were assessed for editing via the knock-down of expression of human Hepatitis B Virus Surface antigen (HBsAg) in a multidose experiment. The human Hepatitis B Virus Surface antigen (HBsAg) ELISA and the total DNA qPCR analysis were performed as described in Example 6.1. [0511] ELISA was performed to measure episomal expression from AAV8-HBV1.2 (SignaGen Laboratories Cat# SL100862) administered as described herein at Day 0 at a dose of 5e10 vg/mouse. At Day 13, serum HBsAg expression was assessed for baseline levels via HBsAg ELISA. Cargos were individually formulated in LNPs, each containing an individual gRNA or mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 729) and another containing an mRNA encoding UGI (open reading frame SEQ ID NO: 732). The LNPs were pre-mixed and administered at a dose level of 0.5 mg/kg (with respect to total RNA cargo content) per body weight of animal at Day 15 and Day 42. The weight ratio of gRNA:BC22n:UGI was 1:2:0.3. Blood was collected at Days 0, 15, 28, 42, and 63, and serum HBsAg expression was determined by ELISA. At Day 84, the animals were euthanized; blood was collected to assess terminal HBsAg and HBV DNA levels in serum. Liver was collected for Next Generation Sequencing (NGS) and editing evaluation at HBV target sites. C-to-T %, C-to-A/G % and indel % were determined by NGS as described in Example 1.1. [0512] HBsAg levels were reduced after the first LNP dose and further reduced for all groups after the second dose. At Day 63 raw data and at Day 84 normalized knockdown showed that multi-dosing has a significant benefit to HBsAg knockdown in 4 out of 5 groups. Example 7 - Dose response curve using Nme2 guides [0513] A dose response transfection of guide RNA was performed to compare potency of selected guides from the gRNA screen (Example 4) to previously identified high activity Nme2 guides targeting HBV genotype D that resulted in HBsAg knockdown. [0514] HepAD38 cells were thawed, passaged, plated, and transfected as described in Example 1.4 with separate lipoplexes formed individually with an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 715), an mRNA encoding UGI (open reading frame SEQ ID NO: 708), or gRNA targeting the integrated S-antigen, Polymerase, or Epsilon locus of HepAD38. [0515] Lipofection reagent was prepared as a mixture of lipids at a ratio of 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG as described in Example 1.2. Single- cargo lipofection reagent was prepared by mixing separately each RNA species: Nme2- BC22n base editor mRNA, UGI mRNA, or gRNA. Each well received 100 ng of base editor and 100 ng of UGI diluted in complete media. A guide RNA dose response curve for each guide tested was formed in complete media with concentrations ranging from 50 nM to 0 nM, titrated using a 3-fold dilution scheme. The media-lipofection reagent mixture was incubated at 37ºC for 10 minutes before addition to HepAD38 cells. The total volume of all three components was 100 µL per well. [0516] Media change, media collection and cell lysis were performed as described in Example 1.4. An MSD assay evaluating HBsAg knockdown in the top two doses of the DRC was performed on collected cell media as described in Example 1.6. Cell lysate was used to quantify intracellular total HBV DNA using a qPCR assay described in Example 1.5. Cell lysates were also prepared for NGS analysis as described in Example 1.1. [0517] The NGS results in Table 27 and Figure 11 show the C-to-T editing outcomes. The EC50 values for the dose response curves in Figure 11 are shown in Table 28. The percent of secreted HBsAg using the top two doses used in the dose response curve is shown in Figure 12 and Table 29.
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Table 29: Percent secreted HBsAg (top two doses of gRNA only) for the extended Nme2 lead guide DRC.
Figure imgf000232_0001
Example 8 - Nme2-BC22n Guides tested in multiplex combinations in HepAD38 [0518] Nme2-BC22n lead guides were tested in multiplex combinations to evaluate combinatorial effects of a multi-guide treatment relative to a single-guide treatment to NGS outcomes as well as HBsAg and total HBV DNA knockdown. [0519] HepAD38 cells were plated in collagen-coated 96-well plates at a density of 20,000 cells/well in complete media (without Doxycycline and G418 Sulfate supplements) as described in Example 1.4. Twenty-four hours later, cells were transfected with lipid nanoparticles (prepared essentially as described in Example 1.2). Lipid nanoparticles used 50% Lipid A, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG by molarity. The lipid nucleic acid assemblies were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a cargo of 1:2:1 for gRNA:Nme2-BC22 (an mRNA encoding Nme2 Cas9 base editor (open reading frame SEQ ID NO: 721)):UGI (mRNA encoding UGI (open reading frame SEQ ID NO: 724)). [0520] A dose response curve was formed in complete media with total LNP cargo masses ranging from 80 ng to 0 ng, titrated using a 2-fold dilution scheme. The media-LNP mixture was incubated at 37ºC for 10 minutes before addition to HepAD38 cells. A total volume of 100 µL/well media-LNP mix was delivered. [0521] Media change, media collection and cell lysis were performed as described in Example 1.4. An MSD assay evaluating HBsAg knockdown was performed on collected cell media as described in Example 1.6. Cell lysates were also prepared for NGS analysis as described in Example 3.2. NGS data is shown in Table 30. Figures 13 and 14 and Table 31 show percent knockdown of secreted HBsAg measured by MSD assay.
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Example 9 - Nme2-BC22n Guides tested in singleplex and multiplex combinations in HBV- infected Primary Human Hepatocytes using lipid nanoparticles [0522] Nme2-BC22n lead guides were tested in singleplex and multiplex combinations in a HBV-infected PHH model. Serially diluted LNP were delivered to evaluate combinatorial effects of a multi-guide treatment relative to a single-guide treatment via secreted HBsAg knockdown (MSD) and total intracellular HBV DNA knockdown (ddPCR). 9.1. Cell culture, infection and LNP delivery [0523] Primary Human Hepatocytes (ThermoFisher, HU8345) were thawed and recovered in CHRMs media (Gibco, CM7000). Cells were resuspended in Primary Human Hepatocyte Plating Media consisting of William’s E media (Gibco, A1217601), hepatocyte plating supplements (Gibco, CM3000) and 5% fetal bovine serum (Gibco, A13450). They were plated in a collagen-coated 96-well plate at a density of 30,000 cells/well. After six hours, plating media was removed and replaced with Primary Hepatocyte Maintenance media consisting of William’s E media, hepatocyte maintenance supplements (Gibco, CM4000), [0524] After one day, the media was removed and replaced with maintenance media containing 2% DMSO for two hours. Media was removed and replaced with HBV inoculum- containing media consisting of maintenance media, 4% PEG-8000, and viral inoculum, MOI 50 (viral inoculum was prepared according to Example 1.5). Uninfected cells received maintenance media with 4% PEG-8000 only. Plates were centrifuged at 1000g for 30 minutes at 37 degrees Celsius before being moved to an incubator. Twenty-four and forty-eight hours later, media was removed and replaced with maintenance media supplemented with 2% DMSO and 2% FBS. [0525] Four days after the previous media change a serially diluted LNP were delivered in maintenance media supplemented with 6% FBS with total cargo masses ranging from 300 ng to 0 ng, titrated using a 10-pt, 3-fold dilution scheme. All LNPs, including a human SCAP targeting control gRNA, were formulated with a cargo ratio of 1:2:0.3 gRNA:Nme2- BC22n:UGI. Multiplex combinations were formed by adding different gRNA LNPs together at a 1:1 ratio prior to serial dilution. The media-LNP mixture was incubated at 37^ for 10 minutes before addition to HBV-infected PHH cells. A total volume of 100 µL/well media-LNP mix was delivered. [0526] Media was replaced with fresh maintenance media supplemented with 2% DMSO and 2% FBS at three days then five days. After the last media change the cells were incubated for five more days and then the media was collected for HBsAg measurement and the cells were lysed using QE buffer for total HBV DNA copy number. 9.2. Measuring secreted HbsAg and intracellular HBV DNA knockdown [0527] Measurement of secreted HbsAg was performed as described in Example 1.6. To determine absolute levels of HbsAg for each sample level, the background subtracted signal from each well was interpolated using a four-parameter non-linear curve of the standard HbsAg protein. To determine relative knockdown of secreted HbsAg the absolute level of each sample was divided by the average of the SCAP control replicates for the same dose of LNP. [0528] Measurement of intracellular HBV DNA copy number was measured by ddPCR (Bio-Rad). Using primers designed against conserved regions of the HBV genome and the Hs Actin Beta (ACTB) gene, a ddPCR reaction was prepared using a ddPCR Supermix for probes (Bio-Rad, 1863024) and readout on the droplet reader following the manufacturer’s protocol. HBV levels for each sample were analyzed by normalizing HBV copy number/uL to ACTB copy number/uL. To determine relative knockdown the housekeeping normalized HBV copy number of each sample was divided by the average of the SCAP control replicates for the same dose of LNP. [0529] Percent HbsAg knockdown data is shown in Table 32 and Figure 15. Percent reduction in HBV DNA copy number is shown in Table 33 and Figure 16. Table 32: Percent HbsAg knockdown with single and multiplexed HBV guides
Figure imgf000252_0001
Figure imgf000253_0001
Table 33: Percent reduction in HBV DNA copy number with single and multiplexed HBV guides
Figure imgf000253_0002
Figure imgf000254_0001
Example 10 – Comparing potent HBV targeting Nme2-BC22n gRNA with different modification patterns in HBV-infected Primary Human Hepatocytes using lipid nanoparticles [0530] Three different Nme2-BC22n lead spacer sequences were tested head-to-head against a different scaffold modification pattern to determine potency differences in HBV- infected PHH. LNP dose-response curves were delivered to evaluate potency via secreted HBsAg knockdown (MSD). [0531] Cell culture, infection, and LNP delivery were performed as in Example 9.1. 10.1. Measuring secreted HBsAg knockdown [0532] Measurement of secreted HBsAg was performed as described in Example 1.6. To determine absolute levels of HBsAg for each sample level, the background subtracted signal from each well was interpolated using a four-parameter non-linear curve of the standard HBsAg protein. gRNA knockdown IC50s (nM gRNA) were calculated from each HBsAg knockdown curve and the IC50 from one modification pattern was compared to the other to determine statistical significance. No statistical differences in HBsAg knockdown potency were noted between the different modification patterns. [0533] HBsAg concentration (ng/ml) is shown in Table 34 and Figure 17. HBsAg IC50 data is in Table 35 and Figure 18. Table 34: HBsAg concentration (ng/mL) following treatment with guides with different modification patterns
Figure imgf000254_0002
Figure imgf000255_0001
Table 35: HBsAg guide IC50 (nM) comparisons
Figure imgf000255_0002
Figure imgf000256_0001
Example 11 - Evaluation of three different Nme2-BC22n gRNA LNP targeting HBV in AAV8-HBV1.2 Mouse Model after two sequential doses of LNP [0534] NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) male mice from Jackson Labs (strain #005557) were dosed at 6-10 weeks of age with AAV8-HBV1.2 Genotype D (Signagen, Cat. # SL100862) and LNP subsequently as indicated, or vehicle (PBS + 0.001% Pluronic for AAV vehicle, TSS for LNP vehicle) via the lateral tail vein. AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein. LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 µl/gram body weight. At various times points post-treatment, blood was collected, and sera was isolated for certain analyses as described further below. 11.1. Human Hepatitis B Virus Surface Antigen (HBsAg) ELISA and Total DNA qPCR analysis [0535] For in vivo studies, blood was collected, and the sera was isolated as indicated. The total human Hepatitis B Virus Surface antigen (HBsAg) levels were determined using an HBsAg (Human) ELISA kit (LifeSpan Bio, Cat. # LS-F37979) according to the manufacturer’s protocol. Serum HBsAg levels were quantitated off a standard curve using 4-parameter logistic fit and expressed as µg/mL of serum. For ddPCR, serum was diluted 1:50 and DNA was isolated using the QE lysis protocol (Lucigen, Cat. # QE09050). ddPCR was run using ddPCR Supermix for probes (Bio-Rad, Cat. #1863024) with a primer set (Fwd-5’- ACTCACCAACCTCTTGTCCT-3’ (SEQ ID NO:X), Rev- 5’- GACAAACGGGCAACATACCT-3’ (SEQ ID NO: X)) against a highly conserved area of the HBV genome not edited by the tested gRNA. A standard plasmid encoding the same HBV sequence was used to determine DNA copies/mL which allowed for a lower limit of linear detection of 2.73e7 copies/mL. [0536] Four sgRNA (one control targeting SCAP, three targeting HBV) were assessed for editing via the knock-down of episomal expression of human Hepatitis B Virus Surface antigen (HBsAg) from AAV8-HBV1.2 (SignaGen Laboratories Cat. # SL100862) administered as described herein at a dose of 1e11 vg/mouse, as measured by ELISA. Two weeks post administration of AAV8-HBV material, serum HBsAg expression was assessed for baseline levels via HBsAg ELISA. LNPs tested in this example were prepared and delivered to mice as described herein three days post the assessment of HBsAg baselines. LNPs, containing gRNA:Nme2-BC22n:UGI at a ratio of 1:2:0.3 were pre-mixed and administered at a dose level of 2 mg/kg (with respect to the total RNA cargo content) per body weight of animal. Thereafter, serum HBsAg expression was determined by ELISA two weeks post the first LNP administration. On the same day, the animals were again administered their respective LNPs at a dose level of 2 mg/kg. [0537] Six weeks post the first LNP dose, the animals were euthanized; blood was collected to assess terminal HBsAg and HBV DNA levels in serum. Phenotypic measurements for each guide-specific target sites are shown in Table 36 and Table 37. Serum HBsAg log10 reduction time course relative to individual animal baseline is shown in Figure 19. Log10 knockdown of total serum HBV DNA relative to TSS group at week 6 is shown in Figure 20. The average HBsAg log10 knockdown from baseline was >2 log10 for G027224 and G030115. All guides resulted in >2 log of serum HBV DNA knockdown but due to assay sensitivity this translated in a maximum detectable knockdown of 2.2 log10. Table 36: Log10 reduction of serum HBsAg relative to baseline
Figure imgf000257_0001
Table 37: Log10 reduction of total serum HBV DNA relative to TSS at week 6
Figure imgf000257_0002
Figure imgf000258_0001
Example 12 - Evaluation of three different Nme2-BC22n gRNA LNP targeting HBV in AAV8-HBV1.2 mouse model comparing editing on HBV A and C genotypes [0538] NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) male mice from Jackson Labs (strain #005557) were dosed at 12 weeks of age with AAV8-HBV1.2 (Signagen, SL100865) Genotype A (AB246338.1) or Genotype C (AB246345.1) and LNP subsequently as indicated, or vehicle (PBS + 0.001% Pluronic for AAV vehicle, TSS for LNP vehicle) via the lateral tail vein. AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein. LNPs were diluted in TSS and administered at amounts as indicated herein, at 10 µl/gram body weight. At various times points post-treatment, blood was collected, and sera was isolated for certain analyses as described further below. 12.1. Human Hepatitis B Virus Surface Antigen (HBsAg) analysis [0539] For in vivo studies, blood was collected, and the sera was isolated as indicated. The total human Hepatitis B Virus Surface antigen (HBsAg) levels were determined using an HBsAg (Human) ELISA kit (LifeSpan Bio, Cat# LS-F37979) according to the manufacturer’s protocol. Serum HBsAg levels were quantitated off a standard curve using 4-parameter logistic fit and expressed as µg/mL of serum. [0540] Four sgRNA (one control targeting Scap, three targeting HBV) were assessed for editing via the knock-down of episomal expression of human Hepatitis B Virus Surface antigen (HBsAg) from AAV8-HBV1.2 Genotype A (AB246338.1) or AAV-HBV1.2 Genotype C (AB246345.1) (SignaGen Laboratories Cat# SL100865) administered as described herein at a dose of 1e11 vg/mouse, as measured by ELISA. G027224 is not fully homologous to the HBV genotypes used in this experiment and has a 1 bp mismatch in guide position 13 relative to Genotype A and a 1 bp mismatich at guide position 16 relative to Genotype C. Two weeks post administration of AAV8-HBV material, serum HBsAg expression was assessed for baseline levels via HBsAg ELISA. LNPs tested in this Example were prepared and delivered to mice as described herein seven days post the assessment of HBsAg baselines. LNPs, containing gRNA:Nme2-BC22n:UGI at a ratio of 1:2:0.3 were pre-mixed and administered at a dose level of 1 mg/kg (with respect to the total RNA cargo content) per body weight of animal. Thereafter, serum HBsAg expression was determined by ELISA two weeks post the first LNP administration. [0541] Four weeks post the first LNP dose, the animals were euthanized; blood was collected to assess terminal HBsAg levels in serum. Serum HBsAg levels and log10 reduction time course relative to individual animal baseline is shown in Figure 21A and 21B and Tables 37 and 38. Endpoint comparisons are described in Figure 22. G030115 and G030120 with perfect spacer matches to the Genotype A and C sequences resulted in significantly better HBsAg knockdown than G027224 (1 bp mismatch) suggesting on-target specificity. Additionally, the rank order of guide performance was maintained across both genotypes and the guides had very similar magnitude HBsAg knockdown in both genotypes although G030115 and G030120 showed slightly greater reductions (significant Welch’s T-test) in secreted HBsAg when targeting Genotype C (Figure 22). Table 37: Log10 serum HBsAg reduction from baseline with HBV genotype A
Figure imgf000259_0001
Table 38: Log10 serum HBsAg reduction from baseline with HBV genotype C
Figure imgf000259_0002
Table 39: Endpoint comparisons of log10 serum HBsAg reduction from baseline
Figure imgf000259_0003
Figure imgf000260_0001
Table 40: Additional Sequences
Figure imgf000260_0002
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001

Claims

We claim: 1. A guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 conitguous nucleotides of nucleotides 163- 466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; B. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 24 contiguous nucleotides of nucleotides 163- 466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; C. a targeting sequence comprising a sequence identical to or complementary to at least 17, 18, preferably 19, or 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1- 843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821- 2460 of SEQ ID NO: 1; or D. a targeting sequence comprising a sequence identical to or complementary to at least 20, 21, 22, preferably 23, or 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1.
2. A guide RNA comprising: A. a targeting sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of: 1. nucleotides 163-466, nucleotides 1-843 , nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; 2. nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; or 3. a target site selected from a target site of any one of HBV number S1-S93 in Table 1A, optionally a target site of any one of HBV number S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 in Table 1A; B. a targeting sequence comprising a sequence identical to or complementary to 17, 18, preferably 19, or 20 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, or to a targeting sequence of S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6 provided in Table 1A; C. a targeting sequence selected from the targeting sequence of G028550, G028551, G028552, G028553, G028554, G028555, G030415, G030418,G030423, G030424, G030426, G030428, G030429, G030430, G030431, G030432, G030435, G030436, G030437, G030439, G030446, G030454, G030456, G030457, G030459, G030460, G030464, G030465, G030468, G030469, G030470, G030471, G030474, G030477, G030478, G030481, G030484, G030485, G030487, G030488, G030489, G030490, G030491, G030492, G030493, G030494, G030496, G030497, G030498, or G030500, optionally G028550, G028554, or G028555; or D. a targeting sequence comprising a sequence identical to or complementary to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460; or nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1.
3. The guide RNA of claim 1 or 2, further comprising one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 268); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 268) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region.
4. The guide RNA of claim 3, wherein the guide RNA lacks 6 nucleotides in shortened hairpin 1.
5. The guide RNA of claim 3, wherein the guide RNA lacks 8 nucleotides in shortened hairpin 1.
6. The guide RNA of any one of claims 3-5, wherein H-1 and H-3 are deleted.
7. The guide RNA of any one of claims 3-6, wherein the guide RNA further comprises a 3’ tail.
8. The guide RNA of claim 7, wherein the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length.
9. The guide RNA of any one of claims 3-8, wherein the guide RNA comprises an upper stem region comprising a modification to any one or more of US1-US12 in the upper stem region.
10. The guide RNA of claim 1 or 2, comprising a nucleotide sequence selected from the sequences in Table 3A.
11. The guide RNA of claim 1 or 2, wherein the guide RNA comprises a modified nucleotide sequence selected from the modified Spy guide scaffold sequences in Table 4, wherein the modified nucleotide sequence is 3’ of the guide sequence.
12. The guide RNA of claim 11, modified according to the pattern of a nucleotide sequence selected from the modified Spy guide RNA sequences in Table 15.
13. The guide RNA of any one of claims 1-12, wherein the guide comprises a nucleotide sequence selected from the unmodified Spy guide RNA Sequences in Table 3B, wherein the N20’s are collectively a targeting sequence of claim 2.
14. The guide RNA of claim 13, wherein each nucleotide of the unmodified Spy guide RNA Sequences in Table 3B is any natural or non-natural nucleotide.
15. The guide RNA of any one of claims 2-10, wherein the guide RNA is modified according to a pattern selected from the modification patterns in Table 15, wherein the (mN*)3N17 refers to the targeting sequence of claim 2 in which the first three nucleotides comprises a 2’-O-Me modification and a phosphorothioate linkage.
16. The guide RNA of claim 1 or 2, comprising a sequence or modification pattern selected from SEQ ID NOs: 269, 273, 274, 362, 366, or 367 as set forth in Table 12.
17. A guide RNA (gRNA) comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of: 1. nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; 2. nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635- 2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330- 353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958- 2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1; or 3. a target site selected from any one of HBV number N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; B. the guide region comprises a nucleic acid sequence identical to or complementary to 21, 22, preferably 23, or 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1; or of a targeting sequence of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37 in Table 2A; C. the guide region is selected from targeting sequence of G027224, G027225, G027228, G030107, G030108, G030115, G030120, G030123, G030125, G030130, G030134, G030138, G030158, G030162, G030167, G030180, or G030181; optionally G027224, G027225, G027228, G030107, G030115, G030120, G030123, G030125, G030130, G030162, or G030167; optionally G027224, G030107, G030115, or G030120; or D. the guide region comprising a sequence identical to or complementary to 24 contiguous nucleotides of a sequence selected from nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, and nucleotides 1821-2460 of SEQ ID NO: 1; or nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1.
18. The guide RNA (gRNA) of claim 17, wherein the conserved region comprises one or more of: (a) a shortened repeat/anti-repeat region, wherein the shortened repeat/anti-repeat region lacks 2-24 nucleotides relative to SEQ ID NO: 455, wherein (i) one or more of nucleotides 37-48 and 53-64 is deleted relative to SEQ ID NO: 455 and optionally one or more of nucleotides 37-64 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 36 is linked to nucleotide 65 by at least 2 nucleotides; or (b) a shortened hairpin 1 region, wherein the shortened hairpin 1 lacks 2-10 nucleotides, optionally 2-8 nucleotides relative to SEQ ID NO: 455 wherein (i) one or more of nucleotides 82-86 and 91-95 is deleted relative to SEQ ID NO: 455 and optionally one or more of positions 82-96 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 81 is linked to nucleotide 96 by at least 4 nucleotides; or (c) a shortened hairpin 2 region, wherein the shortened hairpin 2 lacks 2-18 nucleotides, optionally 2-16 nucleotides relative to SEQ ID NO: 455, wherein (i) one or more of nucleotides 113-121 and 126-134 is deleted relative to SEQ ID NO: 455 and optionally one or more of nucleotides 113-134 is substituted relative to SEQ ID NO: 455; and (ii) nucleotide 112 is linked to nucleotide 135 by at least 4 nucleotides; wherein one or both nucleotides 144-145 are optionally deleted relative to SEQ ID NO: 455; optionally, wherein at least 10 nucleotides are modified nucleotides.
19. The guide RNA of claim 17 or 18, wherein the conserved region comprises a nucleotide sequence selected from Table 5.
20. The guide RNA of any one of claims 1-19, wherein the guide RNA comprises at least one end modification.
21. The guide RNA of claim 20, wherein the modification comprises a 5’ end modification.
22. The guide RNA of claim 20 or 21, wherein the modification comprises a 3’ end modification.
23. The guide RNA of any one of claims 3-22, wherein the guide RNA comprises a modification in a hairpin region.
24. The guide of claim 23, wherein the modification in a hairpin region is also an end modification.
25. The guide RNA of any one of claims 19-24, wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide.
26. The guide RNA of any one of claims 19-25, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides.
27. The guide RNA of any one of claims 19-26, wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide.
28. The guide RNA of any one of claims 19-26, wherein the modification comprises a 2’-fluor (2’F) modified nucleotide.
29. The guide RNA of claim 21, wherein the 5’ end modification comprises a 2’-O- methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide at nucleotides 1-3 of the 5’ end of the guide sequence.
30. The guide RNA of claim 17, wherein the conserved region comprises a modified nucleotide sequence selected from the modified conserved region Nme guide RNA motifs in Table 6, and wherein the conserved region is 3’ of the guide region.
31. The guide RNA of claim 17, wherein the guide RNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 226-234, and wherein the N’s represent the guide sequence of any one of N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37.
32. The guide RNA of claim 31, wherein each nucleotide is any natural or non-natural nucleotide.
33. The guide RNA of claim 17, wherein the guide RNA is modified according to a pattern selected from SEQ ID NOs: 670-678, wherein the N’s are collectively the guide sequence of claim 17, wherein N, A, C, G, and U are ribonucleotides (2’-OH), wherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides.
34. A composition comprising a guide RNA of any one of claims 1-33.
35. A composition comprising a combination of guide RNAs comprising a guide region and a conserved region, wherein: A. the guide region comprises a nucleic acid sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to 24 contiguous nucleotides of target sites selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N37; N24 and N79; N24, N32, and N79; N24, N32, and N37; and N24, N32, and N40; optionally N24 and N37; N7 and N32; N7 and N37; and B. the conserved region comprises a conserved region of any one of claims 18-33.
36. The composition of claim 34 or 35, further comprising an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
37. The composition of claim 36, wherein the nucleic acid encoding the RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding the RNA-guided DNA binding agent.
38. The composition of claim 36 or 37, wherein the RNA-guided DNA binding agent is a nuclease .
39. The composition of any one of claims 36-38, wherein the RNA-guided DNA binding agent is a Cas9 nuclease.
40. The composition of claim 39, wherein the Cas9 is S. pyogenes Cas9.
41. The composition of claim 40, wherein the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 707 and 713 or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to a sequence selected from SEQ ID NOs: 707 and 713.
42. The composition of claim 41, wherein the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706 and 712.
43. The composition of claim 39, wherein the Cas9 is Nme Cas9.
44. The composition of claim 43, wherein Nme Cas9 comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 711, 716, 719, 722, or 730 or an ORF encoding an Nme Cas9 having at least 90% identity to a sequence selected from 600-620.
45. The composition of claim 44, wherein the ORF encoding the amino acid sequence has at least 85% identity to a sequence selected from SEQ ID NOs: 705, 710, 715, 718, 721, or 729.
46. The composition of any one of claims 38-45, wherein the nuclease has double stranded endonuclease activity.
47. The composition of any one of claims 38-45, wherein the nuclease has nickase activity.
48. The composition of any one of claims 38-45, wherein the nuclease is catalytically inactive.
49. The composition of any one of claims 38-48, wherein the nuclease further comprises a heterologous functional domain.
50. The composition of claim 49, wherein the nuclease is a nickase and the heterologous functional domain is a deaminase.
51. The composition of claim 50, wherein the deaminase is a cytidine deaminase or an adenine deaminase.
52. The composition of claim 51, wherein the deaminase is a cytidine deaminase.
53. The composition of claim 52, wherein the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.
54. The composition of claim 53, wherein the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs. 707, 716, 719, 722, or 730or an ORF encoding an amino acid sequence having at least 90% identity to SEQ ID NOs: 707, 716, 719, 722, or 730.
55. The composition of claim 54, wherein the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 706, 715, 718, 721, or 729.
56. The composition of any one of claims 50-55, further comprising a uracil glycosylase inhibitor (UGI) or nucleic acid encoding a UGI, wherein the nuclease polypeptide does not comprise a UGI or the nucleic acid encoding the polypeptide does not encode a UGI.
57. The composition of claim 56, wherein the UGI comprises an amino acid sequence having at least 90% identity to SEQ ID NOs: 709, 725, or 733 or an ORF encoding an amino acid sequence having at least 90% identity to a sequence selected from 709, 725, or 733.
58. The composition of claim 57, wherein the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NO: 708, 724, 732, optionally SEQ ID NO: 708.
59. The composition of any one of claims 37-58, wherein the ORF is a modified ORF.
60. The composition of any one of claims 34-59, further comprising a pharmaceutical excipient.
61. The composition of any one of claims 34-60, wherein the guide RNA is associated with a lipid nanoparticle (LNP).
62. The composition of claim 61, wherein the LNP comprises a cationic lipid.
63. The composition of claim 62, wherein the cationic lipid is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.
64. The composition of any one of claims 61-63, wherein the LNP comprises a helper lipid.
65. The composition of claim 64, wherein the helper lipid is cholesterol.
66. The composition of any one of claims 61-66, wherein the LNP comprises a neutral lipid.
67. The composition of claim 66, wherein the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).
68. The composition of any one of claims 61-67, wherein the LNP comprises a stealth lipid.
69. The composition of claim 68, wherein the stealth lipid is 1,2-dimyristoyl-rac- glycero-3- methoxypolyethylene glycol 2000 (PEG2k-DMG).
70. The composition of claim 61, wherein the LNP comprises (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.
71. A pharmaceutical composition comprising the guide RNA of any one of claims 1- 33 or the composition of any one of claims 34-70.
72. A pharmaceutical composition comprising, or use of, the guide RNA of any one of claims 1-33 or the composition of any one of claims 34-70 for inducing a double stranded break or a single stranded break within an HBV gene in a cell, modifying the nucleic acid sequence of an HBV gene in a cell, or reducing expression of an HBV gene in a cell.
73. The pharmaceutical composition or use of claim 72, wherein the HBV gene is the S gene.
74. The pharmaceutical composition or use of claim 72, for reducing expression of the HBV gene in a cell or subject.
75. A pharmaceutical composition comprising, or use of, the guide RNA of any one of claims 1-33 or the composition of any one of claims 34-70 for treating a subject having hepatitis B.
76. The pharmaceutical composition or use of claim 75, wherein the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the pharmaceutical composition.
77. The pharmaceutical composition or use of claim 75 or 76, for use in combination with an immune stimulator.
78. The pharmaceutical composition or use of claim 77, wherein the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha-2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR-1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1 inhibitor, atezolizumab, avelumab, durvalumab, or an affimer biotherapeutic.
79. The pharmaceutical composition or use of claim 77, wherein the immune stimulator is a vaccine.
80. The pharmaceutical composition or use of claim 79, wherein the vaccine is a nucleic acid vaccine or a protein-based vaccine.
81. The pharmaceutical composition or use of claim 80, wherein the nucleic acid vaccine is an RNA vaccine.
82. The pharmaceutical composition or use of any one of claims 71-82, wherein the subject has chronic HBV.
83. The pharmaceutical composition or use of any one of claims 71-82, wherein the subject is HBe antigen positive.
84. The pharmaceutical composition or use of any one of claims 71-82, wherein the subject is HBe antigen negative.
85. The pharmaceutical composition or use of any one of claims 71-84, wherein the subject has hepatitis D.
86. The pharmaceutical composition or use of claim 85, for treating hepatitis D.
87. A method or inducing a double stranded break or a single stranded break within an HBV gene in a cell or reducing expression of an HBV protein in a cell comprising contacting the cell with the guide RNA of any one of claims 1-33 and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition of any one of claims 34-70.
88. The method of claim 87, wherein the HBV protein is HBsAg protein or HBcrAg protein.
89. The method of claim 87 or 88, wherein the cell is in a subject.
90. The method of claim 87, wherein the HBsAg protein level or the HBcrAg protein level is measured in a subject sample selected from blood or serum.
91. The method of claim 90, wherein the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml.
92. The method of any one of claim 87-91, wherein the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition.
93. The method of any one of claims 87-92, further comprising administration of an immune stimulator.
94. The method of claim 93, wherein the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha- 2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1 inhibitor, atezolizumab, avelumab, durvalumab, or an affimer biotherapeutic.
95. The method of claim 94, wherein the immune stimulator is a vaccine.
96. The method of claim 95, wherein the vaccine is a nucleic acid vaccine or a protein-based vaccine.
97. The method of claim 96, wherein the nucleic acid vaccine is an RNA vaccine.
98. The method of any one of claims 87-97, wherein the subject has chronic HBV.
99. The method of any one of claims 87-97, wherein the subject is HBe antigen positive.
100. The method of any one of claims 87-97, wherein the subject is HBe antigen negative.
101. The method of any one of claims 87-100, wherein the subject has hepatitis D.
102. The method of claim 101, further comprising treating hepatitis D in the subject.
103. The method of any one of claims 87-102, wherein the method comprising administering one or more doses of the guide RNA and the RNA-guided DNA binding agent, of the nucleic acid encoding an RNA-guided DNA binding agent, or of the composition to a subject, optionally comprising administering two, three, four, or five doses.
104. The method of claim 103, comprising administering two or more of the doses, wherein at least two consecutive doses are administered at times separated by at least or about 1 month, 3 months, or 6 months.
105. The method of claim 103, comprising, after administering a first dose, determining HBsAg protein level or HBcrAg protein level in blood or serum of the subject; and subsequently administering to the subject a second dose if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml.
106. The method of claim 105, wherein the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose.
107. Use of the guide RNA of any one of claims 1-33 or the composition of any one of claims 34-70 in the preparation of a medicament for practicing any of the methods of claims 87- 106.
108. A liver cell comprising an HBV genome, wherein the HBV genome comprises a sequence having at least 80%, 85%, optionally 90% or 95% identity, optionally 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245- 264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423- 442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635- 654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1; wherein the HBV genome comprises a genetic modification in the HBV genome within the sequence having at least 80%, 85%, optionally 90%, or 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153- 173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356- 375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621- 640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695- 714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982- 3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1.
109. The liver cell of claim 108, wherein the genetic modification comprises a mutation that generates a stop codon or disrupts initiation of transcription.
110. The liver cell of claim 108, wherein the genetic modification comprises an indel.
111. The liver cell of any one of claims 108-110, wherein the HBV genome comprises a sequence having at least 90%, 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314- 3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817- 1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072- 2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868- 2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1.
112. The liver cell of any one of claims 108-110, wherein the liver cell comprises a second genetic modification in the HBV genome within the sequence having at least 80%, 85%, optionally 90% or 95% identity, or 100% identity to at least 20 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 57-76, 66-85, 126-145, 153-173, 153-172, 154-173, 240-269, 240-259, 245-264, 248-267, 250-269, 269-288, 356-392, 356-375, 361-380, 364-383, 373-392, 423-445, 423-442, 426-445, 574-593, 608-627, 621-655, 621-640, 622-641, 629-648, 629-648, 630-649, 635-654, 636-655, 686-705, 694-716, 694-713, 695-714, 697-716, 713-732, 725-744, 1597-1616, 1817-1836, 1845-1864, 1867-1886, 1880-1903, 1880-1899, 1883-1902, 1884-1903, 2072-2094, 2072-2091, 2073-2092, 2075-2094, 2299-2318, 2348-2367, 2634-2653, 2867-2887, 2867-2886, 2868-2887, 2891-2910, 2956-2975, or 2982-3001; optionally nucleotides 153-173, 153-172, 154-173, or 250-269 of SEQ ID NO: 1.
113. The liver cell of any one of claims 108-112, wherein the genetic modification is within the HBV genome within the sequence having at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity identity to at least 20 contiguous nucleotides of targeting sites in Table 1A, optionally a targeting site of a gRNA with an HBV number selected from S1-S6, S8, S11, S16, S17, S19, S21-25, S28-S30, S32, S39, S47, S49, S50, S52, S53, S57-58, S61-S64, S67, S70, S71, S74, S77, S78, S80-S87, S80, S90-S91, and S93, optionally S1, S5, or S6.
114. The liver cell of any one of claims 108-113, wherein the HBV genome comprises a sequence having 100% identity or at least 80%, 85%, preferably 90%, or 95% identity, or 100% identity to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846, or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146-169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740-763, 755-778, 1390-1413, 1836-1859, 2635- 2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755-778, 1390-1413, 2951-2974, or 2958-2981, optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1.
115. The liver cell of claim 114, wherein the HBV genome comprises a sequence having at least 90% or 95% identity; or 100% identity to at least 24 contiguous nucleotides of nucleotides 163-466, nucleotides 1-843, nucleotides 1-1205, nucleotides 2314-3188, nucleotides 1381-1846 , or nucleotides 1821-2460 of SEQ ID NO: 1, for example nucleotides 35-58, 146- 169, 153-176, 216-239, 330-356, 330-353, 333-356, 342-365, 447-470, 666-689, 713-736, 740- 763, 755-778, 1390-1413, 1836-1859, 2635-2658, 2951-2974, or 2958-2981, optionally nucleotides 146-169, 153-176, 330-356, 330-353, 333-356, 447-470, 713-736, 740-763, 755- 778, 1390-1413, 2951-2974, or 2958-2981 , optionally nucleotides 447-470, 330-353, 755-778, or 713-736 of SEQ ID NO: 1.
116. The liver cell of any one of claims 108-115, wherein the genetic modification is within the sequence having at least 80%, 85%, optionally 90%, or 95% identity or 100% identity to at least 24 contiguous nucleotides of the target sites in Table 2A, optionally a targeting site of a gRNA with an HBV number selected from N7, N8, N11, N24, N25, N32, N37, N40, N42, N47, N51, N55, N75, N79, N84, N97, or N98; optionally N7, N8, N11, N24, N32, N37, N40, N42, N47, N79, or N84; optionally N7, N24, N32, or N37; or a targeting site of gRNAs with a combination of HBV numbers selected from N24 and N37; N7 and N24; N7 and N32; N7 and N37; N7 and N40; N24 and N37; N24 and N40; N37 and N40; N24 and N32; N32 and N37; N24 and N79; N24, N32, and N79; N24, N32, and N37; and N24, N32, and N40; optionally N24 and N37; N7 and N32; N7 and N37.
117. The liver cell of any one of claims 108-116, wherein the HBV genome is integrated into the liver cell genome.
118. The liver cell of any one of claims 108-116, wherein the HBV genome is cccDNA.
119. A method of modifying a nucleic acid sequence of the HBV genome, the method comprising contacting an HBV-infected liver cell the guide RNA of any one of claims 1-33 and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition of any one of claims 34-70.
120. The method of claim 119, wherein the method is performed in vivo, in vitro, or ex vivo.
121. The method of claim 119 or 120, wherein the cell is in a subject infected with HBV.
122. A method of treating an HBV infection in a subject, the method comprising administering to the subject the guide RNA of any one of claims 1-33 and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, or the composition of any one of claims 34-70.
123. The method of any one of claims 119-122, wherein the nucleic acid sequence of the HBV genome encodes an HBV protein.
124. The method of claim 123, wherein the HBV protein is HBsAG or ABcrAg protein.
125. The method of claim 122 or 124, further comprising determining the HBsAg protein level or the HBcrAg protein level in a subject blood or serum sample.
126. The method of claim 125, wherein the serum level of HBsAg is reduced to less than 1000 IU/ml, optionally less than 100 IU/ml.
127. The method of any one of claims 121-126, wherein the subject is not being treated with an agent to reduce serum HBsAg, e.g., nucleotide/ nucleoside inhibitors at the time of administration of the guide or pharmaceutical composition.
128. The method of any one of claims 121-127, further comprising administering an immune stimulator to the subject.
129. The method of claim 128, wherein the immune stimulator is selected from the group PEGylated interferon alpha 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-alpha- 2b, Interferon lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or 9 (TLR3, TLR7, TLR8, TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that inhibits the secretion or release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR- 1221, a cccDNA inhibitor, IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an CTLA-4 inhibitor, ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a PD-L1 inhibitor, atezolizumab, avelumab, durvalumab, or an affimer biotherapeutic.
130. The method of claim 128, wherein the immune stimulator is a vaccine.
131. The method of claim 130, wherein the vaccine is a nucleic acid vaccine or a protein-based vaccine.
132. The method of claim 130, wherein the nucleic acid vaccine is an RNA vaccine.
133. The method of any one of claims 121-132, wherein the subject has chronic HBV.
134. The method of any one of claims 121-133, wherein the subject is HBe antigen positive.
135. The method of any one of claims 121-134, wherein the subject is HBe antigen negative.
136. The method of any one of claims 121-135, wherein the subject has hepatitis D.
137. The method of claim 136, further comprising treating hepatitis D in the subject.
138. The method of any one of claims 119-137, wherein the method comprising administering one or more doses of the guide RNA and the RNA-guided DNA binding agent, of the nucleic acid encoding an RNA-guided DNA binding agent, or of the composition to a subject, optionally comprising administering two, three, four, or five doses..
139. The method of claim 138, comprising administering two or more of the doses, wherein at least two conecutive doses are are administered at times separated by at least or about 1 month, 3 months, or 6 months.
140. The method of claim 138, comprising, after administering a first dose, determining HBsAg protein level or HBcrAg protein level in blood or serum of the subject; and subsequently administering to the subject a second dose if the HBsAg protein level or the HBcrAg protein level is more than 100 IU/ml, 200 IU/ml, 300 IU/ml, 400 IU/ml, 500 IU/ml, 600 IU/ml, 700 IU/ml, 1800 IU/ml, 900 IU/ml, or 1000 IU/ml.
141. The method of any one of claims 121-140, wherein the second dose is administered at least or about 1 month, 3 months, or 6 months after the first dose.
142. Use of the guide RNA of any one of claims 1-33 or the composition of any one of claims 34-70 in the preparation of a medicament for practicing any of the methods of claims 119- 141.
PCT/US2024/018654 2023-03-06 2024-03-06 Compositions and methods for hepatitis b virus (hbv) genome editing WO2024186890A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363450307P 2023-03-06 2023-03-06
US63/450,307 2023-03-06

Publications (1)

Publication Number Publication Date
WO2024186890A1 true WO2024186890A1 (en) 2024-09-12

Family

ID=90717032

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/018654 WO2024186890A1 (en) 2023-03-06 2024-03-06 Compositions and methods for hepatitis b virus (hbv) genome editing

Country Status (1)

Country Link
WO (1) WO2024186890A1 (en)

Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013121A1 (en) 1991-12-24 1993-07-08 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
WO1995032305A1 (en) 1994-05-19 1995-11-30 Dako A/S Pna probes for detection of neisseria gonorrhoeae and chlamydia trachomatis
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US20120276138A1 (en) 2011-03-21 2012-11-01 Tang De-Chu C Rapid and prolonged immunologic-therapeutic
US20130330382A1 (en) 2010-12-31 2013-12-12 Carlton Bradley Brown Fluorocarbon-linked peptide formulation
US20140186958A1 (en) 2012-12-12 2014-07-03 Feng Zhang Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2014152432A2 (en) 2013-03-15 2014-09-25 The General Hospital Corporation Rna-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
CN104498493A (en) * 2014-12-30 2015-04-08 武汉大学 Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA
US9023649B2 (en) 2012-12-17 2015-05-05 President And Fellows Of Harvard College RNA-guided human genome engineering
WO2015089465A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
WO2015089406A1 (en) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Cas variants for gene editing
US20150216967A1 (en) 2010-02-19 2015-08-06 Valneva Austria Gmbh Ic31 nanoparticles
EP2966170A1 (en) * 2014-07-10 2016-01-13 Heinrich-Pette-Institut Leibniz-Institut für experimentelle Virologie-Stiftung bürgerlichen Rechts - HBV inactivation
WO2016010840A1 (en) 2014-07-16 2016-01-21 Novartis Ag Method of encapsulating a nucleic acid in a lipid nanoparticle host
WO2016197132A1 (en) * 2015-06-04 2016-12-08 Protiva Biotherapeutics Inc. Treating hepatitis b virus infection using crispr
US20170114334A1 (en) 2014-06-25 2017-04-27 Caribou Biosciences, Inc. RNA Modification to Engineer Cas9 Activity
WO2017121791A1 (en) 2016-01-12 2017-07-20 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Means and methods for treating hbv
WO2017136794A1 (en) 2016-02-03 2017-08-10 Massachusetts Institute Of Technology Structure-guided chemical modification of guide rna and its applications
WO2017173054A1 (en) 2016-03-30 2017-10-05 Intellia Therapeutics, Inc. Lipid nanoparticle formulations for crispr/cas components
WO2018107028A1 (en) 2016-12-08 2018-06-14 Intellia Therapeutics, Inc. Modified guide rnas
WO2018237369A2 (en) * 2017-06-23 2018-12-27 Vical Incorporated Lipid nanoparticle (lnp)-mediated delivery of a crispr-expressing plasmid dna for treating chronic hepatitis b virus infection
US20190338308A1 (en) 2017-11-10 2019-11-07 University Of Massachusetts Targeted CRISPR Delivery Platforms
WO2019237069A1 (en) 2018-06-08 2019-12-12 Intellia Therapeutics, Inc. Modified guide rnas for gene editing
JP2020055774A (en) * 2018-10-02 2020-04-09 国立大学法人 鹿児島大学 Antiviral drug
WO2020081568A1 (en) 2018-10-15 2020-04-23 University Of Massachusetts Programmable dna base editing by nme2cas9-deaminase fusion proteins
WO2020231863A1 (en) * 2019-05-10 2020-11-19 Beam Therapeutics Inc. Compositions and methods for treating hepatitis b
WO2021119275A1 (en) 2019-12-11 2021-06-17 Intellia Therapeutics, Inc. Modified guide rnas for gene editing
WO2021222287A2 (en) 2020-04-28 2021-11-04 Intellia Therapeutics, Inc. Methods of in vitro cell delivery
EP3957734A1 (en) * 2019-04-18 2022-02-23 Toolgen Incorporated Composition and method for inhibiting proliferation of hepatitis b virus
WO2022261292A1 (en) 2021-06-10 2022-12-15 Intellia Therapeutics, Inc. Modified guide rnas comprising an internal linker for gene editing
WO2024040254A2 (en) * 2022-08-19 2024-02-22 Tune Therapeutics, Inc. Compositions, systems, and methods for regulation of hepatitis b virus through targeted gene repression
WO2024064910A1 (en) * 2022-09-23 2024-03-28 Chroma Medicine, Inc. Compositions and methods for epigenetic regulation of hbv gene expression

Patent Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
WO1993013121A1 (en) 1991-12-24 1993-07-08 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
WO1995032305A1 (en) 1994-05-19 1995-11-30 Dako A/S Pna probes for detection of neisseria gonorrhoeae and chlamydia trachomatis
US20150216967A1 (en) 2010-02-19 2015-08-06 Valneva Austria Gmbh Ic31 nanoparticles
US20130330382A1 (en) 2010-12-31 2013-12-12 Carlton Bradley Brown Fluorocarbon-linked peptide formulation
US20120276138A1 (en) 2011-03-21 2012-11-01 Tang De-Chu C Rapid and prolonged immunologic-therapeutic
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US20140186958A1 (en) 2012-12-12 2014-07-03 Feng Zhang Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US9023649B2 (en) 2012-12-17 2015-05-05 President And Fellows Of Harvard College RNA-guided human genome engineering
WO2014152432A2 (en) 2013-03-15 2014-09-25 The General Hospital Corporation Rna-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
WO2015089465A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
WO2015089406A1 (en) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Cas variants for gene editing
US20150166980A1 (en) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Fusions of cas9 domains and nucleic acid-editing domains
US20170114334A1 (en) 2014-06-25 2017-04-27 Caribou Biosciences, Inc. RNA Modification to Engineer Cas9 Activity
EP2966170A1 (en) * 2014-07-10 2016-01-13 Heinrich-Pette-Institut Leibniz-Institut für experimentelle Virologie-Stiftung bürgerlichen Rechts - HBV inactivation
WO2016010840A1 (en) 2014-07-16 2016-01-21 Novartis Ag Method of encapsulating a nucleic acid in a lipid nanoparticle host
CN104498493A (en) * 2014-12-30 2015-04-08 武汉大学 Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA
WO2016197132A1 (en) * 2015-06-04 2016-12-08 Protiva Biotherapeutics Inc. Treating hepatitis b virus infection using crispr
WO2017121791A1 (en) 2016-01-12 2017-07-20 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Means and methods for treating hbv
WO2017136794A1 (en) 2016-02-03 2017-08-10 Massachusetts Institute Of Technology Structure-guided chemical modification of guide rna and its applications
WO2017173054A1 (en) 2016-03-30 2017-10-05 Intellia Therapeutics, Inc. Lipid nanoparticle formulations for crispr/cas components
WO2018107028A1 (en) 2016-12-08 2018-06-14 Intellia Therapeutics, Inc. Modified guide rnas
WO2018237369A2 (en) * 2017-06-23 2018-12-27 Vical Incorporated Lipid nanoparticle (lnp)-mediated delivery of a crispr-expressing plasmid dna for treating chronic hepatitis b virus infection
US20190338308A1 (en) 2017-11-10 2019-11-07 University Of Massachusetts Targeted CRISPR Delivery Platforms
WO2019237069A1 (en) 2018-06-08 2019-12-12 Intellia Therapeutics, Inc. Modified guide rnas for gene editing
JP2020055774A (en) * 2018-10-02 2020-04-09 国立大学法人 鹿児島大学 Antiviral drug
WO2020081568A1 (en) 2018-10-15 2020-04-23 University Of Massachusetts Programmable dna base editing by nme2cas9-deaminase fusion proteins
EP3957734A1 (en) * 2019-04-18 2022-02-23 Toolgen Incorporated Composition and method for inhibiting proliferation of hepatitis b virus
WO2020231863A1 (en) * 2019-05-10 2020-11-19 Beam Therapeutics Inc. Compositions and methods for treating hepatitis b
WO2021119275A1 (en) 2019-12-11 2021-06-17 Intellia Therapeutics, Inc. Modified guide rnas for gene editing
WO2021222287A2 (en) 2020-04-28 2021-11-04 Intellia Therapeutics, Inc. Methods of in vitro cell delivery
WO2022261292A1 (en) 2021-06-10 2022-12-15 Intellia Therapeutics, Inc. Modified guide rnas comprising an internal linker for gene editing
WO2024040254A2 (en) * 2022-08-19 2024-02-22 Tune Therapeutics, Inc. Compositions, systems, and methods for regulation of hepatitis b virus through targeted gene repression
WO2024064910A1 (en) * 2022-09-23 2024-03-28 Chroma Medicine, Inc. Compositions and methods for epigenetic regulation of hbv gene expression

Non-Patent Citations (45)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. AX741144
"GenBank", Database accession no. GI:21326584
"NCBI", Database accession no. YP _009173870.1
"NCBi", Database accession no. YP_009173867.1
"The Biochemistry of the Nucleic Acids", 1992, pages: 5 - 36
ABBAS ET AL., PROC NATL ACADSCI USA, vol. 114, no. 11, 2017, pages E2016 - E2115
BACKES ET AL., VACCINE, vol. 34, 2016, pages 923 - 32
CARRINGTON ET AL., CELLS, vol. 9, 2020, pages 1690
CONTICELLO ET AL., MOL. BIOL. EVOL., vol. 22, 2005, pages 367 - 77
CONTICELLO, GENOME BIOL, vol. 9, 2008, pages 229
EDRAKI ET AL., MOL. CELL, vol. 73, 2019, pages 1 - 13
ESVELT ET AL., NAT. METH., vol. 10, 2013, pages 1116 - 1121
GENES, vol. 9, no. 10, 2018, pages 495
GILBERT ET AL.: "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes", CELL, vol. 154, 2013, pages 442 - 51, XP055115843, DOI: 10.1016/j.cell.2013.06.044
GREENSAMBROOK: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
GUILINGER J PTHOMPSON D BLIU D R: "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification", NAT. BIOTECHNOL., vol. 32, no. 6, 2014, pages 577 - 82, XP055157221, DOI: 10.1038/nbt.2909
GUO, PMOSS, B, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 4023 - 4027
HOU ET AL., PNAS, vol. 110, 2013, pages 15644 - 15649
HUBER ET AL., J. CELL BIO., vol. 156, 2002, pages 467 - 479
KATIBAH ET AL., PROC NATL ACAD SRI USA, vol. 111, no. 33, 2014, pages 12025 - 30
KIM ET AL., EXP MOL MED., vol. 40, 2008, pages 669 - 676
KIM ET AL., NAT. COMMUN., vol. 8, 2017, pages 145001
KOSTYUSHEV DMITRY ET AL: "Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, BIRKHAUSER VERLAG, HEIDELBERG, DE, vol. 76, no. 9, 23 January 2019 (2019-01-23), pages 1779 - 1794, XP036753035, ISSN: 1420-682X, [retrieved on 20190123], DOI: 10.1007/S00018-019-03021-8 *
LADNER ET AL., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 41, no. 8, 1997, pages 1715 - 1720
LADNER SK ET AL., AAC, vol. 41, 1997, pages 1715 - 1720
LEE ET AL., MOL. THER., vol. 24, 2016, pages 645 - 654
LEGAL ET AL., EMERG. INFECT. DIS., vol. 12, 2006, pages 1447 - 1450
LI ET AL., VACCINE, vol. 33, 2015, pages 4247 - 4254
MAKAROVA ET AL., NAT REV MICROBIOL, vol. 13, no. 11, 2015, pages 722 - 36
MALI ET AL.: "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering", NAT. BIOTECHNOL., vol. 31, 2013, pages 833 - 8, XP055693153, DOI: 10.1038/nbt.2675
MAO, XSHUMAN, S, J. BIOL. CHEM., vol. 269, 1994, pages 24472 - 24479
MURAMATSU ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 18470 - 6
PEREZ-PINERA ET AL.: "RNA-guided gene activation by CRISPR-Cas9-based transcription factors", NAT. METHODS, vol. 10, 2013, pages 973 - 6, XP055181249, DOI: 10.1038/nmeth.2600
QI ET AL.: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression", CELL, vol. 152, 2013, pages 1173 - 83, XP055346792, DOI: 10.1016/j.cell.2013.02.022
SCHELLENBERGER ET AL.: "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner", NAT. BIOTECHNOL., vol. 27, pages 1186 - 1190
SHMAKOV ET AL., MOLECULAR CELL, vol. 60, 2015, pages 385 - 397
STEPINSKI ET AL.: "Synthesis and properties of mRNAs containing the novel 'anti-reverse cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG", RNA, vol. 7, 2001, pages 1486 - 1495, XP002466762
STERNBERG ET AL., NATURE, vol. 520, 2015, pages 186 - 191
VESTERWENGEL, BIOCHEMISTRY, vol. 43, no. 42, 2004, pages 13233 - 41
WHO GUIDELINES FOR PREVENTION, CARE AND TREATMENT OF PERSONS WITH CHRONIC HEPATITIS B INFECTION, 2015, Retrieved from the Internet <URL:apps.who.int/iris/bitstream/handle/10665/154590/9789241549059_eng.pdf>
WI-10 GUIDELINES FOR PREVENTION, CARE AND TREATMENT OF PERSONS WITH CHRONIC HEPATITIS B INFECTION, 2015
ZETSCHE ET AL., CELL, vol. 163, 2015, pages 1 - 13
ZETSCHE ET AL., CELL, vol. 163, no. 3, 22 October 2015 (2015-10-22), pages 759 - 771
ZHEN SHUAI ET AL: "Enhanced antiviral benefit of combination therapy with anti-HBV and anti-PD1 gRNA/cas9 produces a synergistic antiviral effect in HBV infection", MOLECULAR IMMUNOLOGY, PERGAMON, GB, vol. 130, 16 December 2020 (2020-12-16), pages 7 - 13, XP086447013, ISSN: 0161-5890, [retrieved on 20201216], DOI: 10.1016/J.MOLIMM.2020.12.004 *
ZHOU HAO ET AL: "Efficient silencing of hepatitis B virus S gene through CRISPR-mediated base editing", HEPATOLOGY COMMUNICATIONS, vol. 6, no. 7, 26 March 2022 (2022-03-26), pages 1652 - 1663, XP093103623, ISSN: 2471-254X, DOI: 10.1002/hep4.1933 *

Similar Documents

Publication Publication Date Title
Vaillant REP 2139: antiviral mechanisms and applications in achieving functional control of HBV and HDV infection
EP2966170A1 (en) HBV inactivation
De Vreese et al. The molecular target of bicyclams, potent inhibitors of human immunodeficiency virus replication
US20200270617A1 (en) Compositions and methods for transgene expression from an albumin locus
CN108603196A (en) The elimination to mankind&#39;s JC virus and other polyomavirus of guide RNA
US20210163943A1 (en) Compositions and Methods for Hydroxyacid Oxidase 1 (HAO1) Gene Editing for Treating Primary Hyperoxaluria Type 1 (PH1)
KR20210086621A (en) Compositions and methods for lactate dehydrogenase (LDHA) gene editing
US20230295587A1 (en) Compositions and Methods for Kallikrein (KLKB1) Gene Editing
CN112673103B (en) Hepatitis B Virus (HBV) dsRNA agent compositions and methods of use thereof
TW201945388A (en) Optimized engineered meganucleases having specificity for a recognition sequence in the hepatitis B virus genome
US20240002820A1 (en) Polynucleotides, Compositions, and Methods for Genome Editing Involving Deamination
US20170335296A1 (en) Hbv rnase h purification and enzyme inhibitors
CN116745411A (en) Artificial eukaryotic expression system with enhanced performance
US20240067968A1 (en) Compositions, systems, and methods for regulation of hepatitis b virus through targeted gene repression
US20230374456A1 (en) T-Cell Immunoglobulin and Mucin Domain 3 (TIM3) Compositions and Methods for Immunotherapy
Kanagaraj et al. Different antiviral activities of natural APOBEC3C, APOBEC3G, and APOBEC3H variants against hepatitis B virus
JP6986263B2 (en) Antiviral drug
US20230383253A1 (en) Lymphocyte Activation Gene 3 (LAG3) Compositions and Methods for Immunotherapy
JP2023058674A (en) Methods for inhibiting infection and activation of virus
WO2024186890A1 (en) Compositions and methods for hepatitis b virus (hbv) genome editing
TW202436622A (en) Compositions and methods for hepatitis b virus (hbv) genome editing
RU2812046C2 (en) Method for production of arenavirus, as well as arenavirus mutants with antitumor properties
US20240228577A1 (en) Programmed Cell Death Protein 1 (PD1) Compositions and Methods for Cell-Based Therapy
Goguen Optimization of anti-HIV RNAs for use in combination therapy to achieve a functional HIV cure
Bousquet Secreted hepatitis B virus (HBV)-RNA associates with extracellular vesicles in the supernatant of infected hepatocytes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24716044

Country of ref document: EP

Kind code of ref document: A1