US20150284727A1 - Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof - Google Patents

Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof Download PDF

Info

Publication number: US20150284727A1
Authority: US; United States
Prior art keywords: guide rna; composition; cas protein; dna; cas9
Prior art date: 2012-10-23
Legal status (The legal status 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 status listed.): Abandoned

Application number

US14/438,098

Other languages

English (en)

Inventor

Jin-soo Kim

Seung Woo Cho

Sojung Kim

Jong Min Kim

Seokjoong Kim

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Toolgen Inc

Original Assignee

Toolgen 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.)

2012-10-23

Filing date

2013-10-23

Publication date

2015-10-08

Family has litigation

First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=50544909&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20150284727(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.

2013-10-23 Application filed by Toolgen Inc filed Critical Toolgen Inc

2013-10-23 Priority to US14/438,098 priority Critical patent/US20150284727A1/en

2015-05-28 Assigned to TOOLGEN INCORPORATED reassignment TOOLGEN INCORPORATED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHO, SEUNG WOO, KIM, JONG MIN, KIM, SOJUNG, KIM, JIN-SOO, KIM, Seokjoong

2015-10-08 Publication of US20150284727A1 publication Critical patent/US20150284727A1/en

Status Abandoned legal-status Critical Current

Links

Images

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
- C12N15/52—Genes encoding for enzymes or proenzymes
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing 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
- 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
- C12N15/111—General methods applicable to biologically active non-coding 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
- 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
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- 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
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/531—Stem-loop; Hairpin

Definitions

the present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
CRISPRs are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.
CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages.
the CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.
Cas9 an essential protein component in the Type II CRISPR/Cas system, forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements in invading phages or plasmids to protect the host cells.
crRNA is transcribed from the CRISPR element in the host genome, which was previously captured from such foreign invaders.
Jinek et al. (1) demonstrated that a single-chain chimeric RNA produced by fusing an essential portion of crRNA and tracrRNA could replace the two RNAs in the Cas9/RNA complex to form a functional endonuclease.
CRISPR/Cas systems offer an advantage to zinc finger and transcription activator-like effector DNA-binding proteins, as the site specificity in nucleotide binding CRISPR-Cas proteins is governed by a RNA molecule instead of the DNA-binding protein, which can be more challenging to design and synthesize.
RFLP Restriction fragment length polymorphism
Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive T7 endonuclease I (T7E1) or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, Dideoxy sequencing, and deep sequencing.
T7E1 and Surveyor assays are widely used but are cumbersome.
theses enzymes tend to underestimate mutation frequencies because mutant sequences can form homoduplexes with each other and cannot distinguish homozygous bi-allelic mutant clones from wildtype cells.
RFLP is free of these limitations and therefore is a method of choice. Indeed, RFLP was one of the first methods to detect engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction sites. It is possible that no restriction sites are available at the target site of interest.
the present inventors have made many efforts to develop a genome editing method based on CRISPR/Cas system and finally established a programmable RNA-guided endonuclease that cleave DNA in a targeted manner in eukaryotic cells and organisms.
RGENs RNA-guided endonucleases
It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
RNA-guided endonuclease RGEN
the RGEN comprises a guide RNA specific for target DNA and Cas protein.
RGEN RNA-guided endonuclease
It is still another object of the present invention to provide a kit for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method for cleaving a target DNA in eukaryotic cells or organisms comprising a step of transfecting the eukaryotic cells or organisms comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism comprising a step of treating a eukaryotic cell or organism with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
It is still another object of the present invention to provide a method of preparing a genome-modified animal comprising a step of introducing the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
RGEN RNA-guided endonuclease
RGEN RNA-guided endonuclease
the present composition for cleaving a target DNA or inducing a targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, the kit comprising the composition, and the method for inducing targeted mutagenesis provide a new convenient genome editing tools.
custom RGENs can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP, therefore, the composition and method of the present invention may be used in detection and cleaving naturally-occurring variations and mutations.
FIG. 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro.
FIG. 2 shows Cas9-induced mutagenesis at an episomal target site.
(b) Flow cytometry of cells transfected with Cas9. The percentage of cells that express the RFP-GFP fusion protein is indicated.
FIG. 3 shows RGEN-driven mutations at endogenous chromosomal sites.
FIG. 4 shows that RGEN-driven off-target mutations are undetectable.
On-target and potential off-target sequences The human genome was searched in silico for potential off-target sites. Four sites were identified, each of which carries 3-base mismatches with the CCR5 on-target site. Mismatched bases are underlined.
the T7E1 assay was used to investigate whether these sites were mutated in cells transfected with the Cas9/RNA complex. No mutations were detected at these sites. N/A (not applicable), an intergenic site.
Cas9 did not induce off-target-associated chromosomal deletions. The CCR5-specific RGEN and ZFN were expressed in human cells. PCR was used to detect the induction of the 15-kb chromosomal deletions in these cells.
FIG. 5 shows RGEN-induced Foxn1 gene targeting in mice.
(a) A schematic diagram depicting a sgRNA specific to exon 2 of the mouse Foxn1 gene. PAM in exon 2 is shown in red and the sequence in the sgRNA that is complementary to exon 2 is underlined. Triangles indicate cleavage sites.
(b) Representative T7E1 assays demonstrating gene-targeting efficiencies of Cas9 mRNA plus Foxn1-specific sgRNA that were delivered via intra-cytoplasmic injection into one-cell stage mouse embryos. Numbers indicate independent founder mice generated from the highest dose. Arrows indicate bands cleaved by T7E1.
(c) DNA sequences of mutant alleles observed in three Foxn1 mutant founders identified in b.
FIG. 6 shows Foxn1 gene targeting in mouse embryos by intra-cytoplasmic injection of Cas9 mRNA and Foxn1-sgRNA.
FIG. 7 shows Foxn1 gene targeting in mouse embryos using the recombinant Cas9 protein: Foxn1-sgRNA complex.
(a) and (b) are representative T7E1 assays results and their summaries. Embryos were cultivated in vitro after they underwent pronuclear (a) or intra-cytoplasmic injection (b). Numbers in red indicate T7E1-positive mutant founder mice.
FIG. 8 shows Germ-line transmission of the mutant alleles found in Foxn1 mutant founder #12.
FIG. 9 shows Genotypes of embryos generated by crossing Prkdc mutant founders. Prkdc mutant founders ⁇ 25 and ⁇ 15 were crossed and E13.5 embryos were isolated.
FIG. 10 shows Cas9 protein/sgRNA complex induced targeted mutation.
FIG. 11 shows recombinant Cas9 protein-induced mutations in Arabidopsis protoplasts.
FIG. 12 shows recombinant Cas9 protein-induced mutant sequences in the Arabidopsis BRI1 gene.
FIG. 13 shows T7E1 assay showing endogenous CCR5 gene disruption in 293 cells by treatment of Cas9-mal-9R4L and sgRNA/C9R4LC complex.
FIG. 14 ( a, b ) shows mutation frequencies at on-target and off-target sites of RGENs reported in Fu et al. (2013).
T7E1 assays analyzing genomic DNA from K562 cells (R) transfected serially with 20 ⁇ g of Cas9-encoding plasmid and with 60 ⁇ g and 120 ⁇ g of in vitro transcribed GX19 crRNA and tracrRNA, respectively (1 ⁇ 10 6 cells), or (D) co-transfected with 1 ⁇ g of Cas9-encoding plasmid and 1 ⁇ g of GX 19 sgRNA expression plasmid (2 ⁇ 10 5 cells).
FIG. 15 ( a, b ) shows comparison of guide RNA structure. Mutation frequencies of the RGENs reported in Fu et al. (2013) were measured at on-target and off-target sites using the T7E1 assay. K562 cells were co-transfected with the Cas9-encoding plasmid and the plasmid encoding GX19 sgRNA or GGX20 sgRNA. Off-target sites (OT1-3 etc.) are labeled as in Fu et al. (2013).
FIG. 16 shows that in vitro DNA cleavage by Cas9 nickases.
(a) Schematic overview of the Cas9 nuclease and the paired Cas9 nickase. The PAM sequences and cleavage sites are shown in box.
(c) Schematic overview of DNA cleavage reactions. FAM dyes (shown in box) were linked to both 5′ ends of the DNA substrate.
DSBs and SSBs analyzed using fluorescent capillary eletrophoresis. Fluorescentlylabeled DNA substrates were incubated with Cas9 nucleases or nickases before electrophoresis.
FIG. 17 shows comparison of Cas9 nuclease and nickase behavior.
FIG. 18 shows paired Cas9 nickases tested at other endogenous human loci.
(a,c) The sgRNA target sites at human CCR5 and BRCA2 loci. PAM sequences are indicated in red.
(b,d) Genome editing activities at each target site were detected by the T7E1 assay. The repair of two nicks that would produce 5′ overhangs led to the formation of indels much more frequently than did those producing 3′ overhangs.
FIG. 19 shows that paired Cas9 nickases mediate homologous recombination.
FIG. 20 shows DNA splicing induced by paired Cas9 nickases.
FIG. 21 shows that paired Cas9 nickases do not induce translocations.
FIG. 22 shows a conceptual diagram of the T7E1 and RFLP assays.
FIG. 23 shows in vitro cleavage assay of a linearized plasmid containing the C4BPB target site bearing indels.
DNA sequences of individual plasmid substrates (upper panel).
the PAM sequence is underlined. Inserted bases are shown in box.
Arrows (bottom panel) indicate expected positions of DNA bands cleaved by the wild-type-specific RGEN after electrophoresis.
FIG. 24 shows genotyping of mutations induced by engineered nucleases in cells via RGEN-mediated RFLP.
FIG. 25 shows genotyping of RGEN-induced mutations via the RGEN-RFLP technique.
FIG. 26 shows genotyping of mutations induced by engineered nucleases in organisms via RGEN-mediated RFLP.
FIG. 27 shows RGEN-mediated genotyping of ZFN-induced mutations.
the ZFN target site is shown in box.
Black arrows indicate DNA bands cleaved by T7E1.
FIG. 28 shows polymorphic sites in a region of the human HLA-B gene.
the sequence, which surrounds the RGEN target site, is that of a PCR amplicon from HeLa cells. Polymorphic positions are shown in box.
the RGEN target site and the PAM sequence are shown in dashed and bolded box, respectively. Primer sequences are underlined.
FIG. 29 shows genotyping of oncogenic mutations via RGEN-RFLP analysis.
a recurrent mutation c.133-135 deletion of TCT
HCT116 cells was detected by RGENs.
HeLa cells were used as a negative control.
Genotyping of the KRAS substitution mutation c.34 G>A
Mismatched nucleotides are shown in box.
HeLa cells were used as a negative control.
Arrows indicate DNA bands cleaved by RGENs. DNA sequences confirmed by Sanger sequencing are shown.
FIG. 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells via RGEN-RFLP analysis.
FIG. 31 shows genotyping of a KRAS point mutation (c.34 G>A).
Plasmids harboring either the wild-type or mutant KRAS sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
FIG. 32 shows genotyping of a PIK3CA point mutation (c.3140 A>G).
Plasmids harboring either the wild-type or mutant PIK3CA sequences were digested using RGENs with perfectly matched crRNAs or attenuated, one-base mismatched crRNAs. Attenuated crRNAs that were chosen for genotyping are labeled in box above the gels.
FIG. 33 shows genotyping of recurrent point mutations in cancer cell lines.
Genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are shown in box. Black arrows indicate DNA bands cleaved by RGENs.
the present invention provides a composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the present invention provides a use of the composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the composition is also referred to as a RNA-guided endonuclease (RGEN) composition.
RGEN RNA-guided endonuclease
ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequencies obtained with individual nucleases are widely different from each other. Furthermore, some ZFNs and TALENs fail to show any genome editing activities. DNA methylation may limit the binding of these engineered nucleases to target sites. In addition, it is technically challenging and time-consuming to make customized nucleases.
the present inventors have developed a new RNA-guided endonuclease composition based on Cas protein to overcome the disadvantages of ZFNs and TALENs.
an endonuclease activity of Cas proteins has been known. However, it has not been known whether the endonuclease activity of Cas protein would function in an eukaryotic cell because of the complexity of the eukaryotic genome. Further, until now, a composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA specific for the target DNA to cleave a target DNA in eukaryotic cells or organisms has not been developed.
the present RGEN composition based on Cas protein can be more readily customized because only the synthetic guide RNA component is replaced to make a new genome-editing nuclease. No sub-cloning steps are involved to make customized RNA guided endonucleases.
the relatively small size of the Cas gene for example, 4.2 kbp for Cas9 as compared to a pair of TALEN genes ( ⁇ 6 kbp) provides an advantage for this RNA-guided endonuclease composition in some applications such as virus-mediated gene delivery. Further, this RNA-guided endonuclease does not have off-target effects and thus does not induce unwanted mutations, deletion, inversions, and duplications.
RNA-guided endonuclease composition a scalable, versatile, and convenient tools for genome engineering in eukaryotic cells and organisms.
RGEN can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion/deletion (indel) can be analyzed via RGEN-mediated RFLP.
the specificity of RGENs is determined by the RNA component that hybridizes with a target DNA sequence of up to 20 base pairs (bp) in length and by the Cas9 protein that recognize the protospacer-adjacent motif (PAM).
PAM protospacer-adjacent motif
RGENs are readily reprogrammed by replacing the RNA component. Therefore, RGENs provide a platform to use simple and robust RFLP analysis for various sequence variations.
the target DNA may be an endogenous DNA, or artificial DNA, preferably, endogenous DNA.
Cas protein refers to an essential protein component in the CRISPR/Cas system, forms an active endonuclease or nickase when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
crRNA CRISPR RNA
tracrRNA trans-activating crRNA
the information on the gene and protein of Cas are available from GenBank of National Center for Biotechnology Information (NCBI), without limitation.
CRISPR-associated (cas) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. There are three types of CRISPR-Cas system. Among them, Type II CRISPR/Cas system involving Cas9 protein and crRNA and tracrRNA is representative and is well known. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes ( E. coli , Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube).
the Cas protein may be linked to a protein transduction domain.
the protein transduction domain may be poly-arginine or a TAT protein derived from HIV, but it is not limited thereto.
the present composition may comprise Cas component in the form of a protein or in the form of a nucleic acid encoding Cas protein.
Cas protein may be any Cas protein provided that it has an endonuclease or nickase activity when complexed with a guide RNA.
Cas protein is Cas9 protein or variants thereof.
the variant of the Cas9 protein may be a mutant form of Cas9 in which the cataytic asapartate residue is changed to any other amino acid.
the other amino acid may be an alanine, but it is not limited thereto.
Cas protein may be the one isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogens or a recombinant protein, but it is not limited thereto.
the Cas protein derived from Streptococcus pyogens may recognizes NGG trinucleotide.
the Cas protein may comprise an amino acid sequence of SEQ ID NO: 109, but it is not limited thereto.
recombinant when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
a recombinant Cas protein may be generated by reconstituting Cas protein-encoding sequence using the human codon table.
Cas protein-encoding nucleic acid may be a form of vector, such as plasmid comprising Cas-encoding sequence under a promoter such as CMV or CAG.
Cas protein is Cas9
Cas9 encoding sequence may be derived from Streptococcus sp., and preferably derived from Streptococcus pyogenes .
Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID. NO: 1.
Cas9 encoding nucleic acid may comprise the nucleotide sequence having homology of at least 50% to the sequence of SEQ ID NO: 1, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% to the SEQ ID NO:1, but it is not limited thereto.
Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NOs. 108, 110, 106, or 107.
guide RNA refers to a RNA which is specific for the target DNA and can form a complex with Cas protein and bring Cas protein to the target DNA.
the guide RNA may consist of two RNA, i.e., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) or be a single-chain RNA (sgRNA) produced by fusion of an essential portion of crRNA and tracrRNA.
crRNA CRISPR RNA
tracrRNA transactivating crRNA
sgRNA single-chain RNA
the guide RNA may be a dualRNA comprising a crRNA and a tracrRNA.
the guide RNA comprises the essential portion of crRNA and tracrRNA and a portion complementary to a target, any guide RNA may be used in the present invention.
the crRNA may hybridize with a target DNA.
the RGEN may consist of Cas protein, and dualRNA (invariable tracrRNA and target-specific crRNA), or Cas protein and sgRNA (fusion of an essential portion of invariable tracrRNA and target-specific crRNA), and may be readily reprogrammed by replacing crRNA.
the guide RNA further comprises one or more additional nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
the guide RNA further comprises 2-additional guanine nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA.
the guide RNA may be transferred into a cell or an organism in the form of RNA or DNA that encodes the guide RNA.
the guide RNA may be in the form of an isolated RNA, RNA incorporated into a viral vector, or is encoded in a vector.
the vector may be a viral vector, plasmid vector, or agrobacterium vector, but it is not limited thereto.
a DNA that encodes the guide RNA may be a vector comprising a sequence coding for the guide RNA.
the guide RNA may be transferred into a cell or organism by transfecting the cell or organism with the isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
the guide RNA may be transferred into a cell or organism using virus-mediated gene delivery.
the guide RNA When the guide RNA is transfected in the form of an isolated RNA into a cell or organism, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art.
the guide RNA is preferably transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
isolated RNA may be interchangeable to “naked RNA”. This is cost- and time-saving because it does not require a step of cloning.
the use of plasmid DNA or virus-mediated gene delivery for transfection of the guide RNA is not excluded.
the present RGEN composition comprising Cas protein or Cas protein-encoding nucleic acid and a guide RNA can specifically cleave a target DNA due to a specificity of the guide RNA for a target and an endonuclease or nickase activity of Cas protein.
cleavage refers to the breakage of the covalent backbone of a nucleotide molecule.
a guide RNA may be prepared to be specific for any target which is to be cleaved. Therefore, the present RGEN composition can cleave any target DNA by manipulating or genotyping the target-specific portion of the guide RNA.
the guide RNA and the Cas protein may function as a pair.
the term “paired Cas nickase” may refer to the guide RNA and the Cas protein functioning as a pair.
the pair comprises two guide RNAs.
the guide RNA and Cas protein may function as a pair, and induce two nicks on different DNA strand.
the two nicks may be separated by at least 100 bps, but are not limited thereto.
paired Cas nickase allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells.
paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations.
paired nickases did not promote unwanted translocations associated with off-target DNA cleavages.
paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
the composition may be used in the genotyping of a genome in the eukaryotic cells or organisms in vitro.
the guide RNA may comprise the nucleotide sequence of Seq ID. No. 1, wherein the portion of nucleotide position 3 ⁇ 22 is a target-specific portion and thus, the sequence of this portion may be changed depending on a target.
a eukaryotic cell or organism may be yeast, fungus, protozoa, plant, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation.
Cas9 protein/single-chain guide RNA could generate site-specific DNA double-strand breaks in vitro and in mammalian cells, whose spontaneous repair induced targeted genome mutations at high frequencies.
composition comprising Cas protein and a guide RNA may be used to develop therapeutics or value-added crops, livestock, poultry, fish, pets, etc.
the present invention provides a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the present invention provides a use of the composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
the present invention provides a kit for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
the kit may comprise a guide RNA and Cas protein-encoding nucleic acid or Cas protein as separate components or as one composition.
the present kit may comprise some additional components necessary for transferring the guide RNA and Cas component to a cell or an organism.
the kit may comprise an injection buffer such as DEPC-treated injection buffer, and materials necessary for analysis of mutation of a target DNA, but are not limited thereto.
the present invention provides a method for preparing a eukaryotic cell or organism comprising Cas protein and a guide RNA comprising a step of co-transfecting or serial-transfecting the eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein, and a guide RNA or DNA that encodes the guide RNA.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
a Cas protein-encoding nucleic acid or Cas protein and a guide RNA or DNA that encodes the guide RNA may be transferred into a cell by various methods known in the art, such as microinjection, electroporation, DEAEdextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection in protoplast, and so on, but are not limited thereto.
a Cas protein encoding nucleic acid or Cas protein and a guide RNA may be transferred into an organism by various method known in the art to administer a gene or a protein such as injection.
a Cas protein-encoding nucleic acid or Cas protein may be transferred into a cell in the form of complex with a guide RNA, or separately. Cas protein fused to a protein transduction domain such as Tat can also be delivered efficiently into cells.
the eukarotic cell or organisms is co-transfected or serial-transfected with a Cas9 protein and a guide RNA.
serial-transfection may be performed by transfection with Cas protein-encoding nucleic acid first, followed by second transfection with naked guide RNA.
the second transfection is after 3, 6, 12, 18, 24 hours, but it is not limited thereto.
the present invention provides a eukaryotic cell or organism comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the eukaryotic cells or organisms may be prepared by transferring the composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
the eukaryotic cell may be yeast, fungus, protozoa, higher plant, and insect, or amphibian cells, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), graft cells and primary cell culture (in vitro and ex vivo), and in vivo cells, and also mammalian cells including human, which are commonly used in the art, without limitation.
the organism may be yeast, fungus, protozoa, plant, higher plant, insect, amphibian, or mammal.
the present invention provides a method for cleaving a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a step of treating a cell or organism comprising a target DNA with a composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the step of treating a cell or organism with the composition may be performed by transferring the present composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein into the cell or organism.
such transfer may be performed by microinjection, transfection, electroporation, and so on.
the present invention provides an embryo comprising a genome edited by the present RGEN composition comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein.
the embryo may be an embryo of a mouse.
the embryo may be produced by injecting PMSG (Pregnant Mare Serum Gonadotropin) and hCG (human Coldinic Gonadotropin) into a female mouse of 4 to 7 weeks and the super-ovulated female mouse may be mated to males, and the fertilized embryos may be collected from oviduts.
PMSG Pregnant Mare Serum Gonadotropin
hCG human Cold-ovulated female mouse may be mated to males, and the fertilized embryos may be collected from oviduts.
the present RGEN composition introduced into an embryo can cleave a target DNA complementary to the guide RNA by the action of Cas protein and cause a mutation in the target DNA.
the embryo into which the present RGEN composition has been introduced has an edited genome.
the present RGEN composition could cause a mutation in a mouse embryo and the mutation could be transmitted to offsprings.
a method for introducing the RGEN composition into the embryo may be any method known in the art, such as microinjection, stem cell insertion, retrovirus insertion, and so on.
a microinjection technique can be used.
the present invention provides a genome-modified animal obtained by transferring the embryo comprising a genome edited by the present RGEN composition into the oviducts of an animal.
the term “genome-modified animal” refers to an animal of which genome has been modified in the stage of embryo by the present RGEN composition and the type of the animal is not limited.
the genome-modified animal has mutations caused by a targeted mutagenesis based on the present RGEN composition.
the mutations may be any one of deletion, insertion, translocation, inversion.
the site of mutation depends on the sequence of guide RNA of the RGEN composition.
the genome-modified animal having a mutation of a gene may be used to determine the function of the gene.
the present invention provides a method of preparing a genome-modified animal comprising a step of introducing the present RGEN composition comprising a guide RNA specific for the target DNA or DNA that encodes the guide RNA and Cas protein-encoding nucleic acid or Cas protein into an embryo of an animal; and a step of transferring the embryo into a oviduct of pseudopregnant foster mother to produce a genome-modified animal.
the step of introducing the present RGEN composition may be accomplished by any method known in the art such as microinjection, stem cell insertion, retroviral insertion, and so on.
the present invention provides a plant regenerated form the genome-modified protoplasts prepared by the method for eukaryotic cells comprising the RGEN composition.
the present invention provides a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence Cas protein.
the present invention provides a composition for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
genotyping refers to the “Restriction fragment length polymorphism (RFLP) assay”.
RFLP may be used in 1) the detection of indel in cells or organisms induced by the engineered nucleases, 2) the genotyping naturally-occurring mutations or variations in cells or organisms, or 3) the genotyping the DNA of infected pathogenic microorganisms including virus or bacteria, etc.
the mutations or variation may be induced by engineered nucleases in cells.
the engineered nuclease may be a Zinc Finger Nuclease (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), or RGENs, but it is not limited thereto.
ZFNs Zinc Finger Nuclease
TALENs Transcription Activator-Like Effector Nucleases
RGENs RGENs
biological sample includes samples for analysis, such as tissues, cells, whole blood, semm, plasma, saliva, sputum, cerbrospinal fluid or urine, but is not limited thereto
the mutations or variation may be a naturally-occurring mutations or variations.
the mutations or variations are induced by the pathogenic microorganisms. Namely, the mutations or variation occur due to the infection of pathogenic microorganisms, when the pathogenic microorganisms are detected, the biological sample is identified as infected.
the pathogenic microorganisms may be virus or bacteria, but are not limited thereto.
Engineered nuclease-induced mutations are detected by various methods, which include mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescent PCR, DNA melting analysis, and Sanger and deep sequencing.
T7E1 and Surveyor assays are widely used but often underestimate mutation frequencies because the assays detect heteroduplexes (formed by the hybridization of mutant and wild-type sequences or two different mutant sequences); they fail to detect homoduplexes formed by the hybridization of two identical mutant sequences. Thus, these assays cannot distinguish homozygous bialleic mutant clones from wild-type cells nor heterozygous biallelic mutants from heterozygous monoalleic mutants ( FIG. 22 ).
sequence polymorphisms near the nuclease target site can produce confounding results because the enzymes can cleave heteroduplexes formed by hybridization of these different wild-type alleles.
RFLP analysis is free of these limitations and therefore is a method of choice. Indeed, RFLP analysis was one of the first methods used to detect engineered nuclease-mediated mutations. Unfortunately, however, it is limited by the availability of appropriate restriction sites.
the present invention provides a kit for genotyping mutations or variations in an isolated biological sample, comprising the composition for genotyping mutations or variations in an isolated biological sample.
the present invention provides a kit for genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
the present invention provides a method of genotyping mutations or variations in an isolated biological sample, using the composition for genotyping mutations or variations in an isolated biological sample.
the present invention provides a method of genotyping nucleic acid sequences in pathogenic microorganisms in an isolated biological sample, comprising a guide RNA specific for the target DNA sequence and Cas protein.
a guide RNA, Cas protein-encoding nucleic acid or Cas protein are as described in the above.
a Cas9 target sequence consists of a 20-bp DNA sequence complementary to crRNA or a chimeric guide RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas9 itself ( FIG. 1A ).
the Cas9-coding sequence (4,104 bp), derived from Streptococcus pyogenes strain M1 GAS (NC — 002737.1), was reconstituted using the human codon usage table and synthesized using oligonucleotides.
1-kb DNA segments were assembled using overlapping ⁇ 35-mer oligonucleotides and Phusion polymerase (New England Biolabs) and cloned into T-vector (SolGent).
SolGent SolGent
a full-length Cas9 sequence was assembled using four 1-kbp DNA segments by overlap PCR.
the Cas9-encoding DNA segment was subcloned into p3s, which was derived from pcDNA3.1 (Invitrogen).
a peptide tag (NH2-GGSGPPKKKRKVYPYDVPDYA-COOH, SEQ ID NO: 2) containing the HA epitope and a nuclear localization signal (NLS) was added to the C-terminus of Cas9. Expression and nuclear localization of the Cas9 protein in HEK 293T cells were confirmed by western blotting using anti-HA antibody (Santa Cruz).
the Cas9 cassette was subcloned into pET28-b(+) and transformed into BL21(DE3).
the expression of Cas9 was induced using 0.5 mM IPTG for 4 h at 25° C.
the Cas9 protein containing the His6-tag at the C terminus was purified using Ni-NTA agarose resin (Qiagen) and dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol (1).
Cas9 cleaved the plasmid DNA efficiently at the expected position only in the presence of the synthetic RNA and did not cleave a control plasmid that lacked the target sequence ( FIG. 1B ).
a RFP-GFP reporter was used to investigate whether the Cas9/guide RNA complex can cleave the target sequence incorporated between the RFP and GFP sequences in mammalian cells.
the GFP sequence is fused to the RFP sequence out-of-frame (2).
the active GFP is expressed only when the target sequence is cleaved by site-specific nucleases, which causes frameshifting small insertions or deletions (indels) around the target sequence via error-prone non-homologous end-joining (NHEJ) repair of the double-strand break (DSB) ( FIG. 2 ).
the RFP-GFP reporter plasmids used in this study were constructed as described previously (2). Oligonucleotides corresponding to target sites (Table 1) were synthesized (Macrogen) and annealed. The annealed oligonucleotides were ligated into a reporter vector digested with EcoRI and BamHI.
HEK 293T cells were co-transfected with Cas9-encoding plasmid (0.8 ⁇ g) and the RFP-GFP reporter plasmid (0.2 ⁇ g) in a 24-well plate using Lipofectamine 2000 (Invitrogen).
chimeric RNA (1 ⁇ g) prepared by in vitro transcription was transfected using Lipofectamine 2000.
transfected cells were subjected to flow cytometry and cells expressing both RFP and GFP were counted.
GFP-expressing cells were obtained only when the cells were transfected first with the Cas9 plasmid and then with the guide RNA 12 h later ( FIG. 2 ), demonstrating that RGENs could recognize and cleave the target DNA sequence in cultured human cells.
GFP-expressing cells were obtained by serial-transfection of the Cas9 plasmid and the guide RNA rather than co-transfection.
T7E1 T7 endonuclease I
K562 cells were transfected with 20 ⁇ g of Cas9-encoding plasmid using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol.
K562 ATCC, CCL-243 cells were grown in RPMI1640 with 10% FBS and the penicillin/streptomycin mix (100 U/ml and 100 ⁇ g/ml, respectively).
in vitro transcribed chimeric RNA was nucleofected into 1 ⁇ 10 6 K562 cells.
the in vitro transcribed chimeric RNA had been prepared as described in the Example 1-2.
Mutation frequencies (Indels (%) in FIG. 3A ) estimated from the relative DNA band intensities were RNA-dosage dependent, ranging from 1.3% to 5.1%.
DNA sequencing analysis of the PCR amplicons corroborated the induction of RGEN-mediated mutations at the endogenous sites. Indels and microhomologies, characteristic of error-prone NHEJ, were observed at the target site.
the most striking off-target sites associated with these CCR5-specific engineered nucleases reside in the CCR2 locus, a close homolog of CCR5, located 15-kbp upstream of CCR5.
the present inventors intentionally chose the target site of our CCR5-specific RGEN to recognize a region within the CCR5 sequence that has no apparent homology with the CCR2 sequence.
the present inventors investigated whether the CCR5-specific RGEN had off-target effects. To this end, we searched for potential off-target sites in the human genome by identifying sites that are most homologous to the intended 23-bp target sequence. As expected, no such sites were found in the CCR2 gene. Instead, four sites, each of which carries 3-base mismatches with the on-target site, were found ( FIG. 4A ). The T7E1 assays showed that mutations were not detected at these sites (assay sensitivity, ⁇ 0.5%), demonstrating extraordinar specificities of RGENs ( FIG. 4B ).
PCR was used to detect the induction of chromosomal deletions in cells separately transfected with plasmids encoding the ZFN and RGEN specific to CCR5. Whereas the ZFN induced deletions, the RGEN did not ( FIG. 4C ).
RGENs was reprogrammed by replacing the CCR5-specific guide RNA with a newly-synthesized RNA designed to target the human C4BPB gene, which encodes the beta chain of C4b-binding protein, a transcription factor.
RGENs can be delivered into cells in many different forms.
RGENs consist of Cas9 protein, crRNA, and tracrRNA.
the two RNAs can be fused to form a single-chain guide RNA (sgRNA).
sgRNA single-chain guide RNA
a plasmid that encodes Cas9 under a promoter such as CMV or CAG can be transfected into cells.
crRNA, tracrRNA, or sgRNA can also be expressed in cells using plasmids that encode these RNAs.
Use of plasmids however, often results in integration of the whole or part of the plasmids in the host genome.
the bacterial sequences incorporated in plasmid DNA can cause unwanted immune response in vivo.
Plasmid DNA can persist in cells for several days post-transfection, aggravating off-target effects of RGENs.
Recombinant Cas9 protein complexed with in vitro transcribed guide RNA to induce targeted disruption of endogenous genes in human cells.
Recombinant Cas9 protein fused with the hexa-histidine tag was expressed in and purified from E. coli using standard Ni ion affinity chromatography and gel filtration. Purifed recombinant Cas9 protein was concentrated in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol).
Cas9 protein/sgRNA complex was introduced directly into K562 cells by nucleofection: 1 ⁇ 10 6 K562 cells were transfected with 22.5-225 (1.4-14 ⁇ M) of Cas9 protein mixed with 100 ug (29 ⁇ M) of in vitro transcribed sgRNA (or crRNA 40 ug and tracrRNA 80 ug) in 1000 solution using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. After nucleofection, cells were placed in growth media in 6-well plates and incubated for 48 hr.
Cas9 protein/sgRNA complex induced targeted mutation at the CCR5 locus at frequencies that ranged from 4.8 to 38% in a sgRNA or Cas9 protein dose-dependent manner, on par with the frequency obtained with Cas9 plasmid transfection (45%).
Cas9 protein/crRNA/tracrRNA complex was able to induce mutations at a frequency of 9.4%.
Cas9 protein alone failed to induce mutations.
the forkhead box N1 (Foxn1) gene which is important for thymus development and keratinocyte differentiation (Nehls et al., 1996), and the protein kinase, DNA activated, catalytic polypeptide (Prkdc) gene, which encodes an enzyme critical for DNA DSB repair and recombination (Taccioli et al., 1998) were used.
Cas9 mRNA and sgRNAs were synthesized in vitro from linear DNA templates using the mMESSAGE mMACHINE T7 Ultra kit (Ambion) and MEGAshortscript T7 kit (Ambion), respectively, according to the manufacturers' instructions, and were diluted with appropriate amounts of diethyl pyrocarbonate (DEPC, Sigma)-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4). Templates for sgRNA synthesis were generated using oligonucleotides listed in Table 3. Recombinant Cas9 protein was obtained from ToolGen, Inc.
Cas9 mRNA and sgRNAs in M2 medium were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei using a Piezo-driven micromanipulator (Prime Tech).
the recombinant Cas9 protein: Foxn1-sgRNA complex was diluted with DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4) and injected into male pronuclei using a TransferMan NK2 micromanipulator and a FemtoJet microinjector (Eppendorf).
the manipulated embryos were transferred into the oviducts of pseudo-pregnant foster mothers to produce live animals, or were cultivated in vitro for further analyses.
T7E1 assays were performed as previously described using genomic DNA samples from tail biopsies and lysates of whole embryos (Cho et al., 2013).
the genomic region encompassing the RGEN target site was PCR-amplified, melted, and re-annealed to form heteroduplex DNA, which was treated with T7 endonuclease 1 (New England Biolabs), and then analyzed by agarose gel electrophoresis. Potential off-target sites were identified by searching with bowtie 0.12.9 and were also similarly monitored by T7E1 assays.
the primer pairs used in these assays were listed in Tables 4 and 5.
mutant fractions (the number of mutant embryos/the number of total embryos) were dose-dependent, ranging from 33% (1 ng/ ⁇ l sgRNA) to 91% (100 ng/ ⁇ l) ( FIG. 6 b ).
Sequence analysis confirmed mutations in the Foxn1 gene; most mutations were small deletions ( FIG. 6 c ), reminiscent of those induced by ZFNs and TALENs (Kim et al., 2013).
mutant fractions were proportional to the doses of Foxn1-sgRNA, and reached up to 93% (100 ng/ ⁇ l Foxn1-sgRNA) (Tables 6 and 7, FIG. 5 b ).
Prkdc-targeted mice To generate Prkdc-targeted mice, we applied a 5-fold higher concentration of Cas9 mRNA (50 ng/ ⁇ l) with increasing doses of Prkdc-sgRNA (50, 100, and 250 ng/ ⁇ l). Again, the birth rates were very high, ranging from 51% to 60%, enough to produce a sufficient number of newborns for the analysis (Table 6). The mutant fraction was 57% (21 mutant founders among 37 newborns) at the maximum dose of Prkdc-sgRNA. These birth rates obtained with RGENs were approximately 2- to 10-fold higher than those with TALENs reported in our previous study (Sung et al., 2013). These results demonstrate that RGENs are potent gene-targeting reagents with minimal toxicity.
Genotyping Summary Detected alleles 58* 1 not determined ⁇ 11 19 100 bi-allelic ⁇ 60/+1 20 100 bi-allelic ⁇ 67/ ⁇ 19 13 100 bi-allelic ⁇ 18/+455 32 10 bi-allelic (heterozygote) ⁇ 13/ ⁇ 15 + 1 115 10 bi-allelic (heterozygote) ⁇ 18/ ⁇ 5 111 10 bi-allelic (heterozygote) ⁇ 11/+1 110 10 bi-allelic (homozygote) ⁇ 8/ ⁇ 8 120 10 bi-allelic (homozygote) +2/+2 81 100 heterozygote +1/WT 69 100 homozygote ⁇ 11/ ⁇ 11 55 1 mosaic ⁇ 18/ ⁇ 1/ +1/+3 56 1 mosaic ⁇ 127/ ⁇ 41 / ⁇ 2/ +1 127 1 mosaic ⁇ 18 / +1/WT 53 1 mosaic ⁇ 11
the Cas9 coding sequence (4104 bps), derived from Streptococcus pyogenes strain M1 GAS (NC — 002737.1), was cloned to pET28-b(+) plasmid.
a nuclear targeting sequence (NLS) was included at the protein N terminus to ensure the localization of the protein to the nucleus.
pET28-b(+) plasmid containing Cas9 ORF was transformed into BL21(DE3).
Cas9 was then induced using 0.2 mM IPTG for 16 hrs at 18° C. and purified using Ni-NTA agarose beads (Qiagen) following the manufacturer's instructions. Purified Cas9 protein was concentrated using Ultracel—100K (Millipore).
the genomic sequence of the Arabidopsis gene encoding the BRI1 was screened for the presence of a NGG motif, the so called protospacer adjacent motif (PAM), in an exon which is required for Cas9 targeting
PAM protospacer adjacent motif
sgRNAs were produced in vitor using template DNA. Each template DNA was generated by extension with two partially overlapped oligonucleotides (Macrogen, Table X1) and Phusion polymerase (Thermo Scientific) using the following conditions—98° C. 30 sec ⁇ 98° C. 10 sec, 54° C. 20 sec, 72° C. 2 min ⁇ 20, 72° C. 5 min.
Oligonucleotides for the production of the template DNA for in vitro transcription SEQ Oligonuc- ID leotides Sequence (5′-3′) NO BRI1 target 1 GAAATTAATACGACTCACTATAGGTTTGAA 73 (Forward) AGATGGAAGCGCGGGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCG BRI1 target 2 GAAATTAATACGACTCACTATAGGTGAAAC 74 (Forward) TAAACTGGTCCACAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCG Universal AAAAAAGCACCGACTCGGTGCCACTTTTTC 75 (Reverse) AAGTTGATAACGGACTAGCCTTATTTTAAC TTGC
the extended DNA was purified and used as a template for the in vitro production of the guide RNA's using the MEGAshortscript T7 kit (Life Technologies). Guide RNA were then purified by Phenol/Chloroform extraction and ethanol precipitation.
10 ul of purified Cas9 protein (12 ⁇ g/ ⁇ l) and 4 ul each of two sgRNAs (11 ⁇ g/ ⁇ l) were mixed in 20 ⁇ l NEB3 buffer (New England Biolabs) and incubated for 10 min at 37° C.
the leaves of 4-week-old Arabidopsis seedlings grown aseptically in petri dishes were digested in enzyme solution (1% cellulose R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salt) for 8 ⁇ 16 hrs at 25° C. with 40 rpm shaking in the dark. Enzyme/protoplast solutions were filtered and centrifuged at 100 ⁇ g for 3 ⁇ 5 min. Protoplasts were re-suspended in CPW solution after counting cells under the microscope ( ⁇ 100) using a hemacytometer.
enzyme solution 1% cellulose R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salt
protoplasts were re-suspended at 1 ⁇ 10 6 /ml in MMG solution (4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2).
MMG solution 4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2.
Cas9/sgRNA complex 200 ⁇ L (200,000 protoplasts) of the protoplast suspension were gently mixed with 3.3 or 10 uL of Cas9/sgRNA complex [Cas9 protein (6 ⁇ g/ ⁇ L) and two sgRNAs (2.2 ⁇ g/ ⁇ L each)] and 200 ul of 40% polyethylene glycol transfection buffer (40% PEG4000, 200 mM mannitol and 100 mM CaCl2) in 2 ml tubes.
Cas9 with a cysteine at the C-terminal was prepared by PCR amplification using the previously described Cas9 plasmid ⁇ Cho, 2013 #166 ⁇ as the template and cloned into pET28-(a) vector (Novagen, Merk Millipore, Germany) containing His-tag at the N-terminus.
293T Human embryonic kidney cell line
HeLa human ovarian cancer cell line
DMEM Gibco-BRL Rockville
E. coli BL21 cells were transformed with thepET28-(a) vector encoding Cas9 and plated onto Luria-Bertani (LB) agar medium containing 50 ⁇ g/mL kanamycin (Amresco, Solon, Ohio). Next day, a single colony was picked and cultured in LB broth containing 50 ⁇ g/mL kanamycin at 37° C. overnight. Following day, this starter culture at 0.1 OD600 was inoculated into Luria broth containing 50 ⁇ g/mL kanamycin and incubated for 2 hrs at 37° C. until OD600 reached to 0.6-0.8. To induce Cas9 protein expression, the cells were cultured at 30° C. overnight after addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) (Promega, Madison, Wis.) to the final concentration of 0.5 mM.
IPTG isopropyl- ⁇ -D-thiogalactopyranoside
the cells were collected by centrifugation at 4000 rpm for 15-20 mins, resuspendedin a lysis buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, 1 ⁇ protease inhibitor cocktail, 1 mg/ml lysozyme), and lysed by sonication (40% duty, 10 sec pulse, 30 sec rest, for 10 mins on ice).
the soluble fraction was separated as the supernatant after centrifugation at 15,000 rpm for 20 mins at 4° C.
Cas9 protein was purified at 4° C. using a column containing Ni-NTA agarose resin (QIAGEN) and AKTA prime instrument (AKTA prime, GE Healthcare, UK).
soluble protein fractions were loaded onto Ni-NTA agarose resin column (GE Healthcare, UK) at the flow rate of 1 mL/min.
the column was washed with a washing buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 20 mM imidazole, lx protease inhibitor cocktail) and the bound protein was eluted at the flow rate of 0.5 ml/min with an elution buffer (20 mM Tris-Cl pH8.0, 300 mM NaCl, 250 mM imidazole, 1 ⁇ protease inhibitor cocktail).
the pooled eluted fraction was concentrated and dialyzed against storage buffer (50 mM Tris-HCl, pH8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% Glycerol). Protein concentration was quantitated by Bradford assay (Biorad, Hercules, Calif.) and purity was analyzed by SDS-PAGE using bovine serum albumin as the control.
sgRNA (1 ⁇ g) was gently added to various amounts of C9R4LC peptide (ranging from 1 to 40 weight ratio) in 100 ⁇ l of DPBS (pH 7.4). This mixture was incubated at room temperature for 30 mins and diluted to 10 folds using RNAse-free deionized water. The hydrodynamic diameter and z-potential of the formed nanoparticles were measured using dynamic light scattering (Zetasizer-nano analyzer ZS; Malvern instruments, Worcestershire, UK).
Cas9-9R4L and sgRNA-C9R4LC were treated to the cells as follows: 1 ⁇ g of sgRNA and 15 ⁇ g of C9R4LC peptide were added to 250 mL of OPTIMEM medium and incubated at room temperature for 30 mins. At 24 hrs after seeding, cells were washed with OPTIMEM medium and treated with sgRNA-C9R4LC complex for 4 hrs at 37° C. Cells were washed again with OPTIMEM medium and treated with Cas9-9R4L for 2 hrs at 37° C. After treatment, culture media was replaced with serum-containing complete medium and incubated at 37° C. for 24 hrs before the next treatment. Same procedure was followed for multiple treatments of Cas9 and sgRNA for three consecutive days.
Cas9-9R4L and sgRNA-9R4L can Edit Endogenous Genes in Cultured Mammalian Cells without the Use of Additional Delivery Tools
our guide RNA had two additional guanine nucleotides at the 5′ end, which are required for efficient transcription by T7 polymerase in vitro. No such additional nucleotides were included in the sgRNA used by others.
the RNA sequence of our guide RNA can be shown as 5′-GGX 20 , whereas 5′-GX 19 , in which X 20 or GX 19 corresponds to the 20-bp target sequence, represents the sequence used by others.
the first guanine nucleotide is required for transcription by RNA polymerase in cells. To test whether off-target RGEN effects can be attributed to these differences, we chose four RGENs that induced off-target mutations in human cells at high frequencies (13).
SSBs single-strand breaks
HDR homology-directed repair
nickase-induced targeted mutagenesis via HDR is much less efficient than is nuclease-induced mutagenesis.
paired Cas9 nickases would produce composite DSBs, which trigger DNA repair via NHEJ or HDR, leading to efficient mutagenesis ( FIG. 16A ).
paired nickases would double the specificity of Cas9-based genome editing.
Cas9 nucleases and nickases designed to target sites in the AAVS1 locus ( FIG. 16B ) in vitro via fluorescent capillary electrophoresis.
Cas9 nickases composed of guide RNA and a mutant form of Cas9 in which a catalytic aspartate residue is changed to an alanine (D10A Cas9) cleaved only one strand, producing site-specific nicks (FIG. 16 C,D).
some nickases AS1, AS2, AS3, and S6 in FIG.
the Cas9 nuclease complexed with the S2 sgRNA was equally efficient at this site and the on-target site.
D10A Cas9 complexed with the S2 and AS2 sgRNAs discriminated this site from the on-target site by a factor of 270 fold.
This paired nickase also discriminated the AS2 off-target sites (Off-1 and Off-9 in FIG. 17B ) from the on-target site by factors of 160 fold and 990 fold, respectively.
FIG. 21A We then investigated whether Cas9 nucleases and nickases can induce unwanted chromosomal translocations that result from NHEJ repair of on-target and off-target DNA cleavages.
FIG. 21B We were able to detect translocations induced by Cas9 nucleases using PCR (FIG. 21 B,C). No such PCR products were amplified using genomic DNA isolated from cells transfected with the plasmids encoding the AS2+S3 Cas9 nickase pair. This result is in line with the fact that both AS2 and S3 nickases, unlike their corresponding nucleases, did not produce indels at off-target sites ( FIG. 17B ).
paired Cas9 nickases allow targeted mutagenesis and large deletions of up to 1-kbp chromosomal segments in human cells.
paired nickases did not induce indels at off-target sites at which their corresponding nucleases induce mutations.
paired nickases did not promote unwanted translocations associated with off-target DNA cleavages.
paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
paired nickases double the specificity of Cas9-mediated mutagenesis and will broaden the utility of RNA-guided enzymes in applications that require precise genome editing such as gene and cell therapy.
One caveat to this approach is that two highly active sgRNAs are needed to make an efficient nickase pair, limiting targetable sites.
RGENs can be used in Restriction fragment length polymorphism (RFLP) analysis, replacing conventional restriction enzymes.
Engineered nucleases including RGENs induce indels at target sites, when the DSBs caused by the nucleases are repaired by the error-prone non-homologous end-joining (NHEJ) system.
NHEJ error-prone non-homologous end-joining
crRNA and tracrRNA were prepared by in vitro transcription using MEGAshortcript T7 kit (Ambion) according to the manufacturer's instruction. Transcribed RNAs were resolved on a 8% denaturing urea-PAGE gel. The gel slice containing RNA was cut out and transferred to elution buffer. RNA was recovered in nuclease-free water followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry.
Templates for crRNA were prepared by annealing an oligonucleotide whose sequence is shown as 5′-GAAATTAATACGACTCACTATAGGX 20 GTTTTAGAGCTATGCTGTTTTG-3′ (SEQ ID NO: 76), in which X 20 is the target sequence, and its complementary oligonucleotide.
the template for tracrRNA was synthesized by extension of forward and reverse oligonucleotides (5′-GAAATTAATACGACTCACTATAGGAACCATTCAAAACAGCATAGCAAG TTAAAATAAGGCTAGTCCG-3′ (SEQ ID NO: 77) and 5′-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAG CCTTATTTTAACTTGCTATG-3′(SEQ ID NO: 78)) using Phusion polymerase (New England Biolabs).
the Cas9 DNA construct used in our previous Example which encodes Cas9 fused to the His6-tag at the C terminus, was inserted in the pET-28a expression vector.
the recombinant Cas9 protein was expressed in E. coli strain BL21(DE3) cultured in LB medium at 25° C. for 4 hour after induction with 1 mM IPTG. Cells were harvested and resuspended in buffer containing 20 mM Tris PH 8.0, 500 mM NaCl, 5 mM immidazole, and 1 mM PMSF. Cells were frozen in liquid nitrogen, thawed at 4° C., and sonicated.
the Cas9 protein in the lysate was bound to Ni-NTA agarose resin (Qiagen), washed with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 20 mM immidazole, and eluted with buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 250 mM immidazole.
Purified Cas9 protein was dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol and analyzed by SDS-PAGE.
the T7E1 assay was performed as following. In brief, PCR products amplified using genomic DNA were denatured at 95° C., reannealed at 16° C., and incubated with 5 units of T7 Endonuclease I (New England BioLabs) for 20 min at 37° C. The reaction products were resolved using 2 to 2.5% agarose gel electrophoresis.
PCR products (100-150 ng) were incubated for 60 min at 37° C. with optimized concentrations (Table 10) of Cas9 protein, tracrRNA, crRNA in 10 ⁇ l NEB buffer 3 (1 ⁇ ). After the cleavage reaction, RNase A (4 ⁇ g) was added, and the reaction mixture was incubated for 30 min at 37° C. to remove RNA. Reactions were stopped with 6 ⁇ stop solution buffer containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1-2.5% agarose gel electrophoresis and visualized with EtBr staining.
Restriction enzyme-treated linearized plasmid 100 ng was incubated for 60 min at 37° C. with Cas9 protein (0.1 ⁇ g), tracrRNA (60 ng), and crRNA (25 ng) in 10 ⁇ l NEB 3 buffer (1 ⁇ ). Reactions were stopped with 6 ⁇ stop solution containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were resolved with 1% agarose gel electrophoresis and visualized with EtBr staining.
New RGENs with desired DNA specificities can be readily created by replacing crRNA; no de novo purification of custom proteins is required once recombinant Cas9 protein is available.
Engineered nucleases, including RGENs induce small insertions or deletions (indels) at target sites when the DSBs caused by the nucleases are repaired by error-prone non-homologous end-joining (NHEJ).
NHEJ error-prone non-homologous end-joining
RGENs can differentially cleave plasmids that contain wild-type or modified C4BPB target sequences that harbor 1- to 3-base indels at the cleavage site. None of the six plasmids with these indels were cleaved by a C4BPB-specific RGEN5 composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein ( FIG. 23 ). In contrast, the plasmid with the intact target sequence was cleaved efficiently by this RGEN.
Target sequence of RGENs used in this study Gene Target sequence SEQ ID NO human AATGACCACTACATCCTCAA 104 C4BPB GGG mouse Pibf1 AGATGATGTCTCATCATCAG 105 AGG
C4BPB mutant clones used in this study have various mutations ranging from 94 bp deletion to 67 bp insertion ( FIG. 24A ). Importantly, all mutations occurred in mutant clones resulted in the loss of RGEN target site. Among 6 C4BPB clones analyzed, 4 clones have both wildtype and mutant alleles (+/ ⁇ ) and 2 clones have only mutant alleles ( ⁇ / ⁇ ).
PCR products spanning the RGEN target site amplified from wildtype K562 genomic DNA were digested completely by the RGEN composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein expressed in and purified from E. coli (FIG. 24 B/Lane 1).
RGEN target-specific crRNA
tracrRNA tracrRNA
Cas9 protein expressed in and purified from E. coli FIG. 24 B/Lane 1
PCR amplicons of +/ ⁇ clones that contained both wild-type and mutant alleles were partially digested, and those of ⁇ / ⁇ cloned that did not contain the wildtype allele were not digested at all, yielding no cleavage products corresponding to the wildtype sequence ( FIG. 24B ).
RGEN-RFLP analysis is a quantitative method. Genomic DNA samples isolated from the C4BPB null clone and the wild-type cells were mixed at various ratios and used for PCR amplifications. The PCR products were subjected to RGEN genotyping and the T7E1 assay in parallel ( FIG. 25 b ). As expected, DNA cleavage by the RGEN was proportional to the wild type to mutant ratio. In contrast, results of the T7E1 assay correlated poorly with mutation frequencies inferred from the ratios and were inaccurate, especially at high mutant %, a situation in which complementary mutant sequences can hybridize with each other to form homoduplexes.
RGEN genotyping in short
RGEN genotyping was applied to the analysis of mutant mouse founders that had been established by injection of TALENs into mouse one-cell embryos.
FIG. 26A We designed and used an RGEN that recognized the TALEN target site in the Pibf1 gene (Table 10).
Genomic DNA was isolated from a wildtype mouse and mutant mice and subjected to RGEN genotyping after PCR amplification.
RGEN genotyping successfully detected various mutations, which ranged from one to 27-bp deletions ( FIG. 26B ).
RGEN genotyping enabled differential detection of +/ ⁇ and ⁇ / ⁇ founder.
RGENs to detect mutations induced in human cells by a CCR5-specific ZFN, representing yet another class of engineered nucleases ( FIG. 27 ). These results show that RGENs can detect mutations induced by nucleases other than RGENs themselves. In fact, we expect that RGENs can be designed to detect mutations induced by most, if not all, engineered nucleases.
the only limitation in the design of an RGEN genotyping assay is the requirement for the GG or AG (CC or CT on the complementary strand) dinucleotide in the PAM sequence recognized by the Cas9 protein, which occurs once per 4 bp on average.
Indels induced anywhere within the seed region of several bases in crRNA and the PAM nucleotides are expected to disrupt RGEN-catalyzed DNA cleavage. Indeed, we identified at least one RGEN site in most (98%) of the ZFN and TALEN sites.
RGEN-RFLP analysis has applications beyond genotyping of engineered nuclease-induced mutations.
HCT116 human colorectal cancer cell line
PCR products amplified from HCT116 genomic DNA were cleaved partially by both wild-type-specific and mutant-specific RGENs, in line with the heterozygous genotype in HCT116 cells ( FIG. 29 a ).
PCR products amplified from DNA from HeLa cells harboring only wild-type alleles were digested completely by the wild-type-specific RGEN and were not cleaved at all by the mutation-specific RGEN.
HEK293 cells harbor the 32-bp deletion (del32) in the CCR5 gene, which encodes an essential co-receptor of HIV infection: Homozygous del32 CCR5 carriers are immune to HIV infection.
the wild-type-specific RGEN cleaved the PCR products obtained from K562, SKBR3, or HeLa cells (used as wild-type controls) completely but those from HEK293 cells partially ( FIG. 30 a ), confirming the presence of the uncleavable del32 allele in HEK293 cells.
the del32-specific RGEN cleaved the PCR products from wild-type cells as efficiently as those from HEK293 cells.
this RGEN had an off-target site with a single-base mismatch immediately downstream of the on-target site ( FIG. 30 ).
RGENs that contained the perfectly-matched guide RNA specific to the wild-type sequence or mutant sequence cleaved both sequences ( FIGS. 31 a and 32 a ).
RGENs that contained a single-base mismatched guide RNA distinguished the two sequences, enabling genotyping of three recurrent oncogenic point mutations in the KRAS, PIK3CA, and IDH1 genes in human cancer cell lines ( FIG. 29 b and FIGS. 33 a, b ).
RGENs as providing a platform to use simple and robust RFLP analysis for various sequence variations.
RGENs can be used to detect various genetic variations (single nucleotide variations, small insertion/deletions, structural variations) such as diseaserelated recurring mutations, genotypes related to drug-response by a patient and also mutations induced by engineered nucleases in cells.
RGEN genotyping to detect mutations induced by engineered nucleases in cells and animals. In principle, one could also use RGENs that will specifically detect and cleave naturally-occurring variations and mutations.

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Genetics & Genomics (AREA)
Engineering & Computer Science (AREA)
Chemical & Material Sciences (AREA)
Organic Chemistry (AREA)
Zoology (AREA)
Wood Science & Technology (AREA)
Bioinformatics & Cheminformatics (AREA)
Biomedical Technology (AREA)
General Engineering & Computer Science (AREA)
Biotechnology (AREA)
Molecular Biology (AREA)
Biochemistry (AREA)
General Health & Medical Sciences (AREA)
Microbiology (AREA)
Biophysics (AREA)
Physics & Mathematics (AREA)
Plant Pathology (AREA)
Medicinal Chemistry (AREA)
Cell Biology (AREA)
Proteomics, Peptides & Aminoacids (AREA)
Crystallography & Structural Chemistry (AREA)
Analytical Chemistry (AREA)
Immunology (AREA)
Mycology (AREA)
Veterinary Medicine (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)
Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Enzymes And Modification Thereof (AREA)
Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Preparation Of Compounds By Using Micro-Organisms (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Peptides Or Proteins (AREA)

US14/438,098 2012-10-23 2013-10-23 Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof Abandoned US20150284727A1 (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
US14/438,098 US20150284727A1 (en)	2012-10-23	2013-10-23	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

Applications Claiming Priority (5)

Application Number	Priority Date	Filing Date	Title
US201261717324P	2012-10-23	2012-10-23
US201361803599P	2013-03-20	2013-03-20
US201361837481P	2013-06-20	2013-06-20
US14/438,098 US20150284727A1 (en)	2012-10-23	2013-10-23	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
PCT/KR2013/009488 WO2014065596A1 (en)	2012-10-23	2013-10-23	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

Publications (1)

Publication Number	Publication Date
US20150284727A1 true US20150284727A1 (en)	2015-10-08

Family

ID=50544909

Family Applications (9)

Application Number	Title	Priority Date	Filing Date
US14/438,098 Abandoned US20150284727A1 (en)	2012-10-23	2013-10-23	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US14/685,568 Active US10851380B2 (en)	2012-10-23	2015-04-13	Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US14/685,510 Pending US20150344912A1 (en)	2012-10-23	2015-04-13	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,355 Pending US20210047648A1 (en)	2012-10-23	2020-08-27	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,338 Pending US20210047647A1 (en)	2012-10-23	2020-08-27	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/314,050 Pending US20230374525A1 (en)	2012-10-23	2023-05-08	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/313,946 Pending US20230374524A1 (en)	2012-10-23	2023-05-08	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/467,952 Pending US20240240192A9 (en)	2012-10-23	2023-09-15	Cas9/RNA Complexes for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells
US18/467,967 Pending US20240026367A1 (en)	2012-10-23	2023-09-15	Compositions Comprising Cas9/guide RNA Complexes for Inducing Targeted Disruption of Endogenous Genes in Eukaryotic Cells

Family Applications After (8)

Application Number	Title	Priority Date	Filing Date
US14/685,568 Active US10851380B2 (en)	2012-10-23	2015-04-13	Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US14/685,510 Pending US20150344912A1 (en)	2012-10-23	2015-04-13	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,355 Pending US20210047648A1 (en)	2012-10-23	2020-08-27	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US17/004,338 Pending US20210047647A1 (en)	2012-10-23	2020-08-27	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/314,050 Pending US20230374525A1 (en)	2012-10-23	2023-05-08	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/313,946 Pending US20230374524A1 (en)	2012-10-23	2023-05-08	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US18/467,952 Pending US20240240192A9 (en)	2012-10-23	2023-09-15	Cas9/RNA Complexes for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells
US18/467,967 Pending US20240026367A1 (en)	2012-10-23	2023-09-15	Compositions Comprising Cas9/guide RNA Complexes for Inducing Targeted Disruption of Endogenous Genes in Eukaryotic Cells

Country Status (16)

Country	Link
US (9)	US20150284727A1 (zh)
EP (9)	EP4397760A3 (zh)
JP (8)	JP6517143B2 (zh)
KR (5)	KR102182847B1 (zh)
CN (9)	CN110592089B (zh)
AU (5)	AU2013335451C1 (zh)
BR (1)	BR112015008708A2 (zh)
CA (2)	CA2888190C (zh)
DE (2)	DE202013012597U1 (zh)
DK (1)	DK2912175T3 (zh)
ES (3)	ES2886448T3 (zh)
HK (4)	HK1212732A1 (zh)
PL (1)	PL2912175T3 (zh)
PT (1)	PT2912175T (zh)
SG (3)	SG11201503059XA (zh)
WO (1)	WO2014065596A1 (zh)

Cited By (45)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US20150050699A1 (en) *	2012-03-20	2015-02-19	Vilnius University	RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US20150376587A1 (en) *	2014-06-25	2015-12-31	Caribou Biosciences, Inc.	RNA Modification to Engineer Cas9 Activity
US20160002670A1 (en) *	2012-12-17	2016-01-07	President And Fellows Of Harvard College	RNA-Guided Human Genome Engineering
US20160168592A1 (en) *	2013-07-09	2016-06-16	President And Fellows Of Harvard College	Multiplex RNA-Guided Genome Engineering
US20160186208A1 (en) *	2013-04-16	2016-06-30	Whitehead Institute For Biomedical Research	Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
US9677090B2 (en)	2015-10-23	2017-06-13	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US9771600B2 (en)	2015-12-04	2017-09-26	Caribou Biosciences, Inc.	Engineered nucleic acid-targeting nucleic acids
US9856497B2 (en)	2016-01-11	2018-01-02	The Board Of Trustee Of The Leland Stanford Junior University	Chimeric proteins and methods of regulating gene expression
US9888673B2 (en)	2014-12-10	2018-02-13	Regents Of The University Of Minnesota	Genetically modified cells, tissues, and organs for treating disease
US10000772B2 (en)	2012-05-25	2018-06-19	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10166255B2 (en)	2015-07-31	2019-01-01	Regents Of The University Of Minnesota	Intracellular genomic transplant and methods of therapy
US10190137B2 (en)	2013-11-07	2019-01-29	Editas Medicine, Inc.	CRISPR-related methods and compositions with governing gRNAS
WO2019051419A1 (en) *	2017-09-08	2019-03-14	University Of North Texas Health Science Center	MODIFIED CASE VARIANTS9
EP3461894A1 (en)	2015-08-07	2019-04-03	Caribou Biosciences, Inc.	Engineered crispr-cas9 compositions and methods of use
US10286084B2 (en)	2014-02-18	2019-05-14	Duke University	Compositions for the inactivation of virus replication and methods of making and using the same
US10336807B2 (en)	2016-01-11	2019-07-02	The Board Of Trustees Of The Leland Stanford Junior University	Chimeric proteins and methods of immunotherapy
US10428319B2 (en)	2017-06-09	2019-10-01	Editas Medicine, Inc.	Engineered Cas9 nucleases
US10450576B2 (en)	2015-03-27	2019-10-22	E I Du Pont De Nemours And Company	Soybean U6 small nuclear RNA gene promoters and their use in constitutive expression of small RNA genes in plants
US10519457B2 (en)	2013-08-22	2019-12-31	E I Du Pont De Nemours And Company	Soybean U6 polymerase III promoter and methods of use
US20200158716A1 (en) *	2017-07-17	2020-05-21	Massachusetts Institute Of Technology	Cell atlas of healthy and diseased barrier tissues
US10731181B2 (en)	2012-12-06	2020-08-04	Sigma, Aldrich Co. LLC	CRISPR-based genome modification and regulation
US10912797B2 (en)	2016-10-18	2021-02-09	Intima Bioscience, Inc.	Tumor infiltrating lymphocytes and methods of therapy
US20210054371A1 (en) *	2019-08-19	2021-02-25	Minghong Zhong	Conjugates of Guide RNA-Cas Protein Complex
US10934536B2 (en)	2018-12-14	2021-03-02	Pioneer Hi-Bred International, Inc.	CRISPR-CAS systems for genome editing
US20210147879A1 (en) *	2013-11-19	2021-05-20	President And Fellows Of Harvard College	Large Gene Excision and Insertion
US11078481B1 (en)	2016-08-03	2021-08-03	KSQ Therapeutics, Inc.	Methods for screening for cancer targets
US11078483B1 (en)	2016-09-02	2021-08-03	KSQ Therapeutics, Inc.	Methods for measuring and improving CRISPR reagent function
US11098325B2 (en)	2017-06-30	2021-08-24	Intima Bioscience, Inc.	Adeno-associated viral vectors for gene therapy
US11149281B2 (en)	2015-10-06	2021-10-19	Institute For Basic Science	Method for producing genome-modified plants from plant protoplasts at high efficiency
US11236313B2 (en)	2016-04-13	2022-02-01	Editas Medicine, Inc.	Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11312953B2 (en)	2013-03-14	2022-04-26	Caribou Biosciences, Inc.	Compositions and methods of nucleic acid-targeting nucleic acids
US11390884B2 (en)	2015-05-11	2022-07-19	Editas Medicine, Inc.	Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11414657B2 (en)	2015-06-29	2022-08-16	Ionis Pharmaceuticals, Inc.	Modified CRISPR RNA and modified single CRISPR RNA and uses thereof
US11466271B2 (en)	2017-02-06	2022-10-11	Novartis Ag	Compositions and methods for the treatment of hemoglobinopathies
US11499151B2 (en)	2017-04-28	2022-11-15	Editas Medicine, Inc.	Methods and systems for analyzing guide RNA molecules
US11549126B2 (en)	2015-06-03	2023-01-10	Board Of Regents Of The University Of Nebraska	Treatment methods using DNA editing with single-stranded DNA
US11572574B2 (en)	2017-09-28	2023-02-07	Toolgen Incorporated	Artificial genome manipulation for gene expression regulation
US11597924B2 (en)	2016-03-25	2023-03-07	Editas Medicine, Inc.	Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11667911B2 (en)	2015-09-24	2023-06-06	Editas Medicine, Inc.	Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11680268B2 (en)	2014-11-07	2023-06-20	Editas Medicine, Inc.	Methods for improving CRISPR/Cas-mediated genome-editing
US11866726B2 (en)	2017-07-14	2024-01-09	Editas Medicine, Inc.	Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11911415B2 (en)	2015-06-09	2024-02-27	Editas Medicine, Inc.	CRISPR/Cas-related methods and compositions for improving transplantation
US12058986B2 (en)	2017-04-20	2024-08-13	Egenesis, Inc.	Method for generating a genetically modified pig with inactivated porcine endogenous retrovirus (PERV) elements
US12084676B2 (en)	2018-02-23	2024-09-10	Pioneer Hi-Bred International, Inc.	Cas9 orthologs
US12110545B2 (en)	2017-01-06	2024-10-08	Editas Medicine, Inc.	Methods of assessing nuclease cleavage

Families Citing this family (336)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
JP6261500B2 (ja)	2011-07-22	2018-01-17	プレジデントアンドフェローズオブハーバードカレッジ	ヌクレアーゼ切断特異性の評価および改善
US11021737B2 (en)	2011-12-22	2021-06-01	President And Fellows Of Harvard College	Compositions and methods for analyte detection
GB201122458D0 (en)	2011-12-30	2012-02-08	Univ Wageningen	Modified cascade ribonucleoproteins and uses thereof
AU2013251558B2 (en)	2012-04-25	2019-01-03	Regeneron Pharmaceuticals, Inc.	Nuclease-mediated targeting with large targeting vectors
EP3597741A1 (en)	2012-04-27	2020-01-22	Duke University	Genetic correction of mutated genes
CA2877290A1 (en)	2012-06-19	2013-12-27	Daniel F. Voytas	Gene targeting in plants using dna viruses
PL3494997T3 (pl) *	2012-07-25	2020-04-30	The Broad Institute, Inc.	Indukowalne białka wiążące dna i narzędzia perturbacji genomu oraz ich zastosowania
WO2014065596A1 (en)	2012-10-23	2014-05-01	Toolgen Incorporated	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US8697359B1 (en) *	2012-12-12	2014-04-15	The Broad Institute, Inc.	CRISPR-Cas systems and methods for altering expression of gene products
WO2014093655A2 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
IL307735A (en) *	2012-12-12	2023-12-01	Broad Inst Inc	Systems engineering, methods and optimal guiding components for sequence manipulation
WO2014093694A1 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
CA2894684A1 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Engineering and optimization of improved crispr-cas systems, methods and enzyme compositions for sequence manipulation in eukaryotes
EP2931892B1 (en)	2012-12-12	2018-09-12	The Broad Institute, Inc.	Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
JP2016505256A (ja) *	2012-12-12	2016-02-25	ザ・ブロード・インスティテュート・インコーポレイテッ	配列操作のためのＣＲＩＳＰＲ−Ｃａｓ成分系、方法および組成物
RU2721275C2 (ru)	2012-12-12	2020-05-18	Те Брод Инститьют, Инк.	Доставка, конструирование и оптимизация систем, способов и композиций для манипуляции с последовательностями и применения в терапии
EP2921557B1 (en)	2012-12-12	2016-07-13	The Broad Institute, Inc.	Engineering of systems, methods and optimized guide compositions for sequence manipulation
EP3578666A1 (en)	2013-03-12	2019-12-11	President and Fellows of Harvard College	Method of generating a three-dimensional nucleic acid containing matrix
CN105209624A (zh) *	2013-03-15	2015-12-30	明尼苏达大学董事会	采用CRISPR/Cas系统的植物基因组的工程改造
WO2014165825A2 (en) *	2013-04-04	2014-10-09	President And Fellows Of Harvard College	Therapeutic uses of genome editing with crispr/cas systems
PL3456831T3 (pl)	2013-04-16	2021-12-06	Regeneron Pharmaceuticals, Inc.	Ukierunkowana modyfikacja genomu szczura
WO2014186686A2 (en) *	2013-05-17	2014-11-20	Two Blades Foundation	Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases
ES2670531T3 (es) *	2013-05-29	2018-05-30	Cellectis S.A.	Un método para producir una escisión de ADN precisa utilizando la actividad nickasa de Cas9
US20140356956A1 (en)	2013-06-04	2014-12-04	President And Fellows Of Harvard College	RNA-Guided Transcriptional Regulation
CA3176690A1 (en) *	2013-06-04	2014-12-11	President And Fellows Of Harvard College	Rna-guided transcriptional regulation
AU2014279694B2 (en)	2013-06-14	2020-07-23	Cellectis	Methods for non-transgenic genome editing in plants
EP3725885A1 (en)	2013-06-17	2020-10-21	The Broad Institute, Inc.	Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof
CN105492611A (zh)	2013-06-17	2016-04-13	布罗德研究所有限公司	用于序列操纵的优化的crispr-cas双切口酶系统、方法以及组合物
EP3011029B1 (en)	2013-06-17	2019-12-11	The Broad Institute, Inc.	Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
DK3011031T3 (da)	2013-06-17	2020-12-21	Broad Inst Inc	Fremføring og anvendelse af crispr-cas-systemerne, vektorer og sammensætninger til levermålretning og -terapi
JP6702858B2 (ja)	2013-06-17	2020-06-03	ザ・ブロード・インスティテュート・インコーポレイテッド	ウイルス成分を使用して障害および疾患をターゲティングするためのＣＲＩＳＰＲ−Ｃａｓ系および組成物の送達、使用および治療上の適用
JP2016528890A (ja)	2013-07-09	2016-09-23	プレジデントアンドフェローズオブハーバードカレッジ	ＣＲＩＳＰＲ／Ｃａｓ系を用いるゲノム編集の治療用の使用
US10563225B2 (en)	2013-07-26	2020-02-18	President And Fellows Of Harvard College	Genome engineering
US9163284B2 (en)	2013-08-09	2015-10-20	President And Fellows Of Harvard College	Methods for identifying a target site of a Cas9 nuclease
SG11201601927SA (en)	2013-08-16	2016-04-28	Massachusetts Inst Technology	Selective delivery of material to cells
US9359599B2 (en)	2013-08-22	2016-06-07	President And Fellows Of Harvard College	Engineered transcription activator-like effector (TALE) domains and uses thereof
US9388430B2 (en)	2013-09-06	2016-07-12	President And Fellows Of Harvard College	Cas9-recombinase fusion proteins and uses thereof
US9228207B2 (en)	2013-09-06	2016-01-05	President And Fellows Of Harvard College	Switchable gRNAs comprising aptamers
US9737604B2 (en)	2013-09-06	2017-08-22	President And Fellows Of Harvard College	Use of cationic lipids to deliver CAS9
ES2681622T3 (es)	2013-09-18	2018-09-14	Kymab Limited	Métodos, células y organismos
WO2015065964A1 (en)	2013-10-28	2015-05-07	The Broad Institute Inc.	Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof
US10584358B2 (en)	2013-10-30	2020-03-10	North Carolina State University	Compositions and methods related to a type-II CRISPR-Cas system in Lactobacillus buchneri
KR101773782B1 (ko)	2013-12-11	2017-09-01	리제너론 파마슈티칼스 인코포레이티드	게놈의 표적화된 변형을 위한 방법 및 조성물
SI3080279T1 (sl)	2013-12-11	2019-01-31	Regeneron Pharmaceuticals, Inc.	Postopki in sestavki za ciljano spremembo genoma
EP3080271B1 (en)	2013-12-12	2020-02-12	The Broad Institute, Inc.	Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems
WO2015089364A1 (en)	2013-12-12	2015-06-18	The Broad Institute Inc.	Crystal structure of a crispr-cas system, and uses thereof
US9840699B2 (en)	2013-12-12	2017-12-12	President And Fellows Of Harvard College	Methods for nucleic acid editing
CN106103705A (zh)	2013-12-12	2016-11-09	布罗德研究所有限公司	核苷酸重复障碍中crispr‑cas系统的组合物和使用方法
JP2017503485A (ja)	2013-12-12	2017-02-02	ザ・ブロード・インスティテュート・インコーポレイテッド	遺伝子産物の発現、構造情報、及び誘導性モジュラーｃａｓ酵素を変更するためのｃｒｉｓｐｒ−ｃａｓ系並びに方法
SG10201804976YA (en)	2013-12-12	2018-07-30	Broad Inst Inc	Delivery, Use and Therapeutic Applications of the Crispr-Cas Systems and Compositions for Genome Editing
US10787654B2 (en)	2014-01-24	2020-09-29	North Carolina State University	Methods and compositions for sequence guiding Cas9 targeting
AU2015217208B2 (en)	2014-02-11	2018-08-30	The Regents Of The University Of Colorado, A Body Corporate	CRISPR enabled multiplexed genome engineering
ES2898460T3 (es)	2014-02-27	2022-03-07	Monsanto Technology Llc	Composiciones y procedimientos para la modificación genómica dirigida al sitio
EP3957735A1 (en)	2014-03-05	2022-02-23	Editas Medicine, Inc.	Crispr/cas-related methods and compositions for treating usher syndrome and retinitis pigmentosa
US11141493B2 (en)	2014-03-10	2021-10-12	Editas Medicine, Inc.	Compositions and methods for treating CEP290-associated disease
EP3116997B1 (en)	2014-03-10	2019-05-15	Editas Medicine, Inc.	Crispr/cas-related methods and compositions for treating leber's congenital amaurosis 10 (lca10)
US11339437B2 (en)	2014-03-10	2022-05-24	Editas Medicine, Inc.	Compositions and methods for treating CEP290-associated disease
WO2015148863A2 (en)	2014-03-26	2015-10-01	Editas Medicine, Inc.	Crispr/cas-related methods and compositions for treating sickle cell disease
CA2944978C (en)	2014-04-08	2024-02-13	North Carolina State University	Methods and compositions for rna-directed repression of transcription using crispr-associated genes
JP2017517256A (ja) *	2014-05-20	2017-06-29	リージェンツオブザユニバーシティオブミネソタ	遺伝子配列を編集する方法
KR101886381B1 (ko) *	2014-05-28	2018-08-10	기초과학연구원	불활성화된 표적 특이적 뉴클레아제를 이용한 표적 dna의 분리 방법
US9970001B2 (en)	2014-06-05	2018-05-15	Sangamo Therapeutics, Inc.	Methods and compositions for nuclease design
EP3708671A1 (en)	2014-06-06	2020-09-16	Regeneron Pharmaceuticals, Inc.	Methods and compositions for modifying a targeted locus
WO2015191693A2 (en)	2014-06-10	2015-12-17	Massachusetts Institute Of Technology	Method for gene editing
WO2015192020A1 (en)	2014-06-13	2015-12-17	Children's Medical Center Corporation	Products and methods to isolate mitochondria
LT3354732T (lt)	2014-06-23	2020-04-10	Regeneron Pharmaceuticals, Inc.	Nukleazės tarpininkaujamas dnr surinkimas
WO2015200555A2 (en) *	2014-06-25	2015-12-30	Caribou Biosciences, Inc.	Rna modification to engineer cas9 activity
LT3161128T (lt)	2014-06-26	2018-10-25	Regeneron Pharmaceuticals, Inc.	Būdai ir kompozicijos, skirti tikslinei genetinei modifikacijai, ir panaudojimo būdai
US10676754B2 (en)	2014-07-11	2020-06-09	E I Du Pont De Nemours And Company	Compositions and methods for producing plants resistant to glyphosate herbicide
EP3169776A4 (en) *	2014-07-14	2018-07-04	The Regents of The University of California	Crispr/cas transcriptional modulation
AU2015294354B2 (en) *	2014-07-21	2021-10-28	Illumina, Inc.	Polynucleotide enrichment using CRISPR-Cas systems
EP4079847A1 (en)	2014-07-30	2022-10-26	President And Fellows Of Harvard College	Cas9 proteins including ligand-dependent inteins
CN113789317B (zh)	2014-08-06	2024-02-23	基因工具股份有限公司	使用空肠弯曲杆菌crispr/cas系统衍生的rna引导的工程化核酸酶的基因编辑
EP3194578B1 (en) *	2014-08-06	2021-03-10	College of Medicine Pochon Cha University Industry-Academic Cooperation Foundation	Immune-compatible cells created by nuclease-mediated editing of genes encoding hla
US11071289B2 (en)	2014-08-14	2021-07-27	Biocytogen Boston Corp	DNA knock-in system
US9970030B2 (en)	2014-08-27	2018-05-15	Caribou Biosciences, Inc.	Methods for increasing CAS9-mediated engineering efficiency
EP3633032A3 (en)	2014-08-28	2020-07-29	North Carolina State University	Novel cas9 proteins and guiding features for dna targeting and genome editing
WO2016036754A1 (en)	2014-09-02	2016-03-10	The Regents Of The University Of California	Methods and compositions for rna-directed target dna modification
US11560568B2 (en)	2014-09-12	2023-01-24	E. I. Du Pont De Nemours And Company	Generation of site-specific-integration sites for complex trait loci in corn and soybean, and methods of use
CN104212836A (zh) *	2014-09-18	2014-12-17	东华大学	一种在哺乳动物细胞系中敲除mir-505的方法
SG10201913797YA (en)	2014-10-15	2020-03-30	Regeneron Pharma	Methods and compositions for generating or maintaining pluripotent cells
CN107109487B (zh)	2014-10-15	2022-03-04	赛琪科学股份有限公司	自动处理核酸和电泳样品制备的装置、方法和系统
CN107109362A (zh)	2014-10-31	2017-08-29	麻省理工学院	递送生物分子至免疫细胞
US10208298B2 (en) *	2014-11-06	2019-02-19	E.I. Du Pont De Nemours And Company	Peptide-mediated delivery of RNA-guided endonuclease into cells
US11352666B2 (en)	2014-11-14	2022-06-07	Institute For Basic Science	Method for detecting off-target sites of programmable nucleases in a genome
JP6621820B2 (ja) *	2014-11-14	2019-12-18	インスティチュートフォーベーシックサイエンスＩｎｓｔｉｔｕｔｅＦｏｒＢａｓｉｃＳｃｉｅｎｃｅ	ゲノムでプログラマブルヌクレアーゼの非標的位置を検出する方法
EP3521437A1 (en)	2014-11-21	2019-08-07	Regeneron Pharmaceuticals, Inc.	Methods and compositions for targeted genetic modification using paired guide rnas
JP7068821B2 (ja)	2014-12-03	2022-05-17	アジレント・テクノロジーズ・インク	化学修飾を有するガイドｒｎａ
EP3230451B1 (en)	2014-12-12	2021-04-07	The Broad Institute, Inc.	Protected guide rnas (pgrnas)
AU2015362784B2 (en)	2014-12-16	2021-05-13	Danisco Us Inc	Fungal genome modification systems and methods of use
WO2016100951A2 (en) *	2014-12-18	2016-06-23	Integrated Dna Technologies, Inc.	Crispr-based compositions and methods of use
ES2947714T3 (es)	2014-12-19	2023-08-17	Regeneron Pharma	Métodos y composiciones para la modificación genética dirigida a través de múltiples direccionamientos en un solo paso
EP3234111A1 (en)	2014-12-19	2017-10-25	Regeneron Pharmaceuticals, Inc.	Stem cells for modeling type 2 diabetes
WO2016115179A1 (en) *	2015-01-12	2016-07-21	Massachusetts Institute Of Technology	Gene editing through microfluidic delivery
UA125246C2 (uk) *	2015-03-16	2022-02-09	Інстітьют Оф Генетікс Енд Дівелопментл Байолоджі, Чайніз Екадемі Оф Сайнсис	Спосіб здійснення сайт-спрямованої модифікації рослинних геномів із застосуванням неуспадковуваних матеріалів
EP3271461A1 (en) *	2015-03-20	2018-01-24	Danmarks Tekniske Universitet	Crispr/cas9 based engineering of actinomycetal genomes
EP3851530A1 (en) *	2015-03-26	2021-07-21	Editas Medicine, Inc.	Crispr/cas-mediated gene conversion
JP6873911B2 (ja)	2015-04-06	2021-05-19	ザボードオブトラスティーズオブザレランドスタンフォードジュニアユニバーシティー	初代細胞において標的核酸の遺伝子調節を誘導するためにインビトロで行う方法
AU2016253150B2 (en)	2015-04-24	2022-04-21	Editas Medicine, Inc.	Evaluation of Cas9 molecule/guide RNA molecule complexes
AU2016261600B2 (en)	2015-05-08	2021-09-23	President And Fellows Of Harvard College	Universal donor stem cells and related methods
KR101785847B1 (ko)	2015-05-12	2017-10-17	연세대학교 산학협력단	선형 이중가닥 ＤＮＡ를 활용한 ＣＲＩＳＰＲ／Ｃａｓ９ 시스템을 이용한 표적 유전체 교정
EP3095870A1 (en)	2015-05-19	2016-11-23	Kws Saat Se	Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable therefrom
KR20220139447A (ko)	2015-05-29	2022-10-14	노쓰 캐롤라이나 스테이트 유니버시티	크리스퍼 핵산을 사용하여 박테리아, 고세균, 조류 및 효모를 스크리닝하는 방법
CA2983874C (en)	2015-06-15	2022-06-21	North Carolina State University	Methods and compositions for efficient delivery of nucleic acids and rna-based antimicrobials
TW202400626A (zh) *	2015-06-18	2024-01-01	美商博得學院股份有限公司	降低脫靶效應的ｃｒｉｓｐｒ酶突變
WO2016205759A1 (en)	2015-06-18	2016-12-22	The Broad Institute Inc.	Engineering and optimization of systems, methods, enzymes and guide scaffolds of cas9 orthologs and variants for sequence manipulation
US10648020B2 (en)	2015-06-18	2020-05-12	The Broad Institute, Inc.	CRISPR enzymes and systems
JP6925984B2 (ja)	2015-07-09	2021-08-25	マサチューセッツインスティテュートオブテクノロジー	無核細胞への物質の送達
EP3329001B1 (en) *	2015-07-28	2021-09-22	Danisco US Inc.	Genome editing systems and methods of use
CA3033909A1 (en)	2015-08-14	2017-02-23	The University Of Sydney	Connexin 45 inhibition for therapy
CA2996001A1 (en) *	2015-08-25	2017-03-02	Duke University	Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases
US11613759B2 (en)	2015-09-04	2023-03-28	Sqz Biotechnologies Company	Intracellular delivery of biomolecules to cells comprising a cell wall
US20180237800A1 (en) *	2015-09-21	2018-08-23	The Regents Of The University Of California	Compositions and methods for target nucleic acid modification
EP3356533A1 (en)	2015-09-28	2018-08-08	North Carolina State University	Methods and compositions for sequence specific antimicrobials
US11970710B2 (en)	2015-10-13	2024-04-30	Duke University	Genome engineering with Type I CRISPR systems in eukaryotic cells
CA2996329A1 (en) *	2015-10-20	2017-04-27	Pioneer Hi-Bred International, Inc.	Restoring function to a non-functional gene product via guided cas systems and methods of use
EP3365356B1 (en)	2015-10-23	2023-06-28	President and Fellows of Harvard College	Nucleobase editors and uses thereof
AU2016349288A1 (en)	2015-11-03	2018-05-31	President And Fellows Of Harvard College	Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix
KR101885901B1 (ko) *	2015-11-13	2018-08-07	기초과학연구원	5' 말단의 인산기가 제거된 rna를 포함하는 리보핵산단백질 전달용 조성물
CN108699101B (zh) *	2015-11-20	2022-04-19	塞奇科学股份有限公司	用于基因组dna片段的靶向纯化的制备型电泳方法
CN106811479B (zh) *	2015-11-30	2019-10-25	中国农业科学院作物科学研究所	利用CRISPR/Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用
US20170151287A1 (en)	2015-11-30	2017-06-01	Flagship Ventures Management, Inc.	Methods and compositions of chondrisomes
CN106845151B (zh) *	2015-12-07	2019-03-26	中国农业大学	CRISPR-Cas9系统sgRNA作用靶点的筛选方法及装置
WO2017099494A1 (ko) *	2015-12-08	2017-06-15	기초과학연구원	Cpf1을 포함하는 유전체 교정용 조성물 및 그 용도
US11542466B2 (en)	2015-12-22	2023-01-03	North Carolina State University	Methods and compositions for delivery of CRISPR based antimicrobials
EP3402327A1 (en) *	2016-01-15	2018-11-21	The Jackson Laboratory	Genetically modified non-human mammals by multi-cycle electroporation of cas9 protein
US11512311B2 (en)	2016-03-25	2022-11-29	Editas Medicine, Inc.	Systems and methods for treating alpha 1-antitrypsin (A1AT) deficiency
WO2017173054A1 (en) *	2016-03-30	2017-10-05	Intellia Therapeutics, Inc.	Lipid nanoparticle formulations for crispr/cas components
CN116200465A (zh)	2016-04-25	2023-06-02	哈佛学院董事及会员团体	用于原位分子检测的杂交链反应方法
CN107326046A (zh) *	2016-04-28	2017-11-07	上海邦耀生物科技有限公司	一种提高外源基因同源重组效率的方法
SG10202102885UA (en)	2016-05-20	2021-05-28	Regeneron Pharma	Methods for breaking immunological tolerance using multiple guide rnas
CA3025523A1 (en) *	2016-05-27	2017-11-30	Aadigen, Llc	Peptides and nanoparticles for intracellular delivery of genome-editing molecules
LT3604527T (lt)	2016-06-02	2021-06-25	Sigma-Aldrich Co., Llc	Programuojamų, dnr surišančių baltymų panaudojimas tiksliniam genomo modifikavimui pagerinti
US10767175B2 (en)	2016-06-08	2020-09-08	Agilent Technologies, Inc.	High specificity genome editing using chemically modified guide RNAs
US20190177710A1 (en) *	2016-06-15	2019-06-13	Toolgen Incorporated	Method For Screening Target Specific Nuclease Using Multi-Target System Of On-Target And Off-Target And Use Thereof
US20190330603A1 (en) *	2016-06-17	2019-10-31	Genesis Technologies Limited	Crispr-cas system, materials and methods
KR20190019168A (ko) *	2016-06-17	2019-02-26	더 브로드 인스티튜트, 인코퍼레이티드	제vi형 crispr 오솔로그 및 시스템
DK3472325T3 (da) *	2016-06-20	2024-05-21	Keygene Nv	Fremgangsmåde til målrettet DNA-ændring i planteceller
DK3474669T3 (da)	2016-06-24	2022-06-27	Univ Colorado Regents	Fremgangsmåde til generering af stregkodede kombinatoriske biblioteker
WO2018009525A1 (en) *	2016-07-05	2018-01-11	The Johnson Hopkins University	Crispr/cas9-based compositions and methods for treating cancer
EP3484994A4 (en) *	2016-07-13	2020-01-22	DSM IP Assets B.V.	CRISPR-CAS-SYSTEM FOR AN ALGENE CELL
US20190309283A1 (en) *	2016-07-19	2019-10-10	Biodynamics Laboratory, Inc.	Method for preparing long-chain single-stranded dna
US11123409B2 (en)	2016-07-28	2021-09-21	Institute For Basic Science	Method of treating or preventing eye disease using Cas9 protein and guide RNA
JP2019523009A (ja)	2016-07-29	2019-08-22	リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎＰｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ．	Ｃ末端切断型フィブリリン−１の発現をもたらす変異を有するマウス
BR112019001887A2 (pt)	2016-08-02	2019-07-09	Editas Medicine Inc	composições e métodos para o tratamento de doença associada a cep290
WO2018027078A1 (en)	2016-08-03	2018-02-08	President And Fellows Of Harard College	Adenosine nucleobase editors and uses thereof
AU2017308889B2 (en)	2016-08-09	2023-11-09	President And Fellows Of Harvard College	Programmable Cas9-recombinase fusion proteins and uses thereof
KR101710026B1 (ko)	2016-08-10	2017-02-27	주식회사 무진메디	Cas9 단백질 및 가이드 RNA의 혼성체를 함유하는 나노 리포좀 전달체 조성물
KR101856345B1 (ko) *	2016-08-24	2018-06-20	경상대학교산학협력단	CRISPR/Cas9 시스템을 이용하여 APOBEC3H 및 APOBEC3CH 이중-넉아웃 고양이를 제조하는 방법
WO2018039438A1 (en)	2016-08-24	2018-03-01	President And Fellows Of Harvard College	Incorporation of unnatural amino acids into proteins using base editing
US20190225974A1 (en)	2016-09-23	2019-07-25	BASF Agricultural Solutions Seed US LLC	Targeted genome optimization in plants
US10717978B2 (en)	2016-10-07	2020-07-21	Integrated Dna Technologies, Inc.	S. pyogenes CAS9 mutant genes and polypeptides encoded by same
US11242542B2 (en)	2016-10-07	2022-02-08	Integrated Dna Technologies, Inc.	S. pyogenes Cas9 mutant genes and polypeptides encoded by same
KR101997116B1 (ko)	2016-10-14	2019-07-05	연세대학교 산학협력단	Kras 유전자에 상보적인 가이드 rna 및 이의 용도
WO2018071868A1 (en)	2016-10-14	2018-04-19	President And Fellows Of Harvard College	Aav delivery of nucleobase editors
WO2018144097A1 (en)	2016-11-04	2018-08-09	Akeagen Llc	Genetically modified non-human animals and methods for producing heavy chain-only antibodies
US20190264218A1 (en)	2016-11-04	2019-08-29	Flagship Pioneering Innovations V, Inc.	Novel Plant Cells, Plants, and Seeds
EP3896162A1 (en) *	2016-11-14	2021-10-20	Toolgen Incorporated	Artificially engineered sc function control system
EP3545085A4 (en)	2016-11-22	2020-10-28	Integrated Dna Technologies, Inc.	CRISPR / CPF1 SYSTEMS AND METHODS
US11293022B2 (en)	2016-12-12	2022-04-05	Integrated Dna Technologies, Inc.	Genome editing enhancement
KR101748575B1 (ko) *	2016-12-16	2017-06-20	주식회사 엠젠플러스	Ins 유전자 녹아웃 당뇨병 또는 당뇨병 합병증 동물모델 및 이의 제조방법
US20200208166A1 (en) *	2016-12-22	2020-07-02	Toolgen Incorporated	Oleic Acid-Enriched Plant Body Having Genetically Modified FAD2 And Production Method Thereof
US10745677B2 (en)	2016-12-23	2020-08-18	President And Fellows Of Harvard College	Editing of CCR5 receptor gene to protect against HIV infection
CN110300802A (zh) *	2016-12-23	2019-10-01	基础科学研究院	用于动物胚胎碱基编辑的组合物和碱基编辑方法
JP6994730B2 (ja) *	2016-12-26	2022-02-04	独立行政法人酒類総合研究所	ゲノム編集タンパク質の直接導入による糸状菌ゲノム編集方法
US11859219B1 (en)	2016-12-30	2024-01-02	Flagship Pioneering Innovations V, Inc.	Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
KR102515727B1 (ko) *	2017-01-11	2023-03-30	주식회사 툴젠	중첩된 가이드핵산을 이용한 표적 핵산에 특정 핵산 서열을 삽입하기 위한 조성물 및 방법
KR20190111067A (ko)	2017-01-23	2019-10-01	리제너론 파마슈티칼스 인코포레이티드	하이드록시스테로이드 17-베타 탈수소효소 13 (hsd17b13) 변이체 및 이의 용도
AU2018212986B2 (en)	2017-01-28	2024-07-25	Inari Agriculture Technology, Inc.	Novel plant cells, plants, and seeds
CN111051509A (zh) *	2017-03-06	2020-04-21	基础科学研究院	用于电介质校准的含有c2cl核酸内切酶的组合物以及使用其进行电介质校准的方法
WO2018165504A1 (en)	2017-03-09	2018-09-13	President And Fellows Of Harvard College	Suppression of pain by gene editing
JP2020510439A (ja)	2017-03-10	2020-04-09	プレジデントアンドフェローズオブハーバードカレッジ	シトシンからグアニンへの塩基編集因子
WO2018170184A1 (en)	2017-03-14	2018-09-20	Editas Medicine, Inc.	Systems and methods for the treatment of hemoglobinopathies
JP7191388B2 (ja)	2017-03-23	2022-12-19	プレジデントアンドフェローズオブハーバードカレッジ	核酸によってプログラム可能なｄｎａ結合蛋白質を含む核酸塩基編集因子
AU2018250330A1 (en)	2017-04-07	2019-09-19	Sage Science, Inc.	Systems and methods for detection of genetic structural variation using integrated electrophoretic DNA purification
US11767512B2 (en)	2017-04-13	2023-09-26	Cellectis	Sequence specific reagents targeting CCR5 in primary hematopoietic cells
US11913015B2 (en)	2017-04-17	2024-02-27	University Of Maryland, College Park	Embryonic cell cultures and methods of using the same
JP2020518253A (ja) *	2017-05-05	2020-06-25	インスティテュート・オブ・ジェネティクス・アンド・ディヴェロプメンタル・バイオロジー、チャイニーズ・アカデミー・オブ・サイエンシズＩｎｓｔｉｔｕｔｅｏｆＧｅｎｅｔｉｃｓａｎｄＤｅｖｅｌｏｐｍｅｎｔａｌＢｉｏｌｏｇｙ，ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＳｃｉｅｎｃｅｓ	トランスジェニックマーカー配列を使用せず細胞を単離する方法
EP3622070A2 (en)	2017-05-10	2020-03-18	Editas Medicine, Inc.	Crispr/rna-guided nuclease systems and methods
WO2018209320A1 (en)	2017-05-12	2018-11-15	President And Fellows Of Harvard College	Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation
EP3635102A1 (en)	2017-06-05	2020-04-15	Regeneron Pharmaceuticals, Inc.	B4galt1 variants and uses thereof
CN108977442B (zh) *	2017-06-05	2023-01-06	广州市锐博生物科技有限公司	用于dna编辑的系统及其应用
CN111108208A (zh)	2017-06-13	2020-05-05	旗舰先锋创新V股份有限公司	包含愈合子的组合物及其用途
KR20240027153A (ko)	2017-06-15	2024-02-29	더 리전트 오브 더 유니버시티 오브 캘리포니아	표적화된 비-바이러스 dna 삽입
US9982279B1 (en)	2017-06-23	2018-05-29	Inscripta, Inc.	Nucleic acid-guided nucleases
US10011849B1 (en)	2017-06-23	2018-07-03	Inscripta, Inc.	Nucleic acid-guided nucleases
CN107488710B (zh) *	2017-07-14	2020-09-22	上海吐露港生物科技有限公司	一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒
US11732274B2 (en)	2017-07-28	2023-08-22	President And Fellows Of Harvard College	Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
CA3065579A1 (en)	2017-07-31	2019-02-07	Regeneron Pharmaceuticals, Inc.	Assessment of crispr/cas-induced recombination with an exogenous donor nucleic acid in vivo
KR20240145526A (ko)	2017-07-31	2024-10-07	리제너론 파마슈티칼스 인코포레이티드	Cas-형질전환 마우스 배아 줄기 세포 및 마우스 및 이것의 용도
WO2019028023A2 (en)	2017-07-31	2019-02-07	Regeneron Pharmaceuticals, Inc.	METHODS AND COMPOSITIONS FOR EVALUATING CRISPR / CAS MEDIATED DISRUPTION OR EXCISION AND CRISPR / CAS INDUCED RECOMBINATION USING IN VIVO EXOGENIC DONOR NUCLEIC ACID
EP3663310A4 (en)	2017-08-04	2021-08-11	Peking University	TALE-RVD WITH SPECIFIC DETECTION OF A DNA BASE MODIFIED BY METHYLATION AND APPLICATION OF IT
CN111278983A (zh)	2017-08-08	2020-06-12	北京大学	基因敲除方法
EP3676376A2 (en)	2017-08-30	2020-07-08	President and Fellows of Harvard College	High efficiency base editors comprising gam
CN111194350A (zh)	2017-09-18	2020-05-22	博雅辑因（北京）生物科技有限公司	一种基因编辑t细胞及其用途
BR112020006428A2 (pt) *	2017-09-28	2020-09-24	Toolgen Incorporated	manipulação de genoma artificial para regulação de expressão de genes
AU2018339089A1 (en) *	2017-09-29	2020-04-09	Intellia Therapeutics, Inc.	Polynucleotides, compositions, and methods for genome editing
KR102207352B1 (ko) *	2017-09-29	2021-01-26	서울대학교산학협력단	Klotho 유전자 넉아웃 질환모델 동물 및 이의 용도
NZ761329A (en)	2017-09-29	2023-04-28	Regeneron Pharma	Non-human animals comprising a humanized ttr locus and methods of use
AU2018352592A1 (en)	2017-10-16	2020-06-04	Beam Therapeutics, Inc.	Uses of adenosine base editors
EA202091056A1 (ru)	2017-10-27	2020-09-17	Те Риджентс Оф Те Юниверсити Оф Калифорния	Направленная замена эндогенных т-клеточных рецепторов
CN109722414A (zh)	2017-10-27	2019-05-07	博雅辑因（北京）生物科技有限公司	一种高效制备成熟红细胞的方法以及用于制备成熟红细胞的培养基
CN108192956B (zh) *	2017-11-17	2021-06-01	东南大学	一种基于Cas9核酸酶的DNA检测分析方法及其应用
CN111836894B (zh)	2017-11-21	2023-11-10	基恩科雷有限责任公司	用于使用CRISPR/Cpf1系统进行基因组编辑的组合物及其用途
WO2019138083A1 (en)	2018-01-12	2019-07-18	Basf Se	Gene underlying the number of spikelets per spike qtl in wheat on chromosome 7a
CN111742051A (zh) *	2018-01-23	2020-10-02	基础科学研究院	延伸的单向导rna及其用途
US11926835B1 (en)	2018-01-29	2024-03-12	Inari Agriculture Technology, Inc.	Methods for efficient tomato genome editing
KR102086689B1 (ko) *	2018-02-06	2020-03-09	주식회사 진씨커	돌연변이 세포 유리 유전자 분리 키트 및 이를 이용한 돌연변이 세포 유리 유전자 분리 방법
WO2019156475A1 (ko) *	2018-02-06	2019-08-15	주식회사 진씨커	돌연변이 세포 유리 유전자 분리 키트 및 이를 이용한 돌연변이 세포 유리 유전자 분리 방법
JP2021518102A (ja)	2018-03-14	2021-08-02	エディタス・メディシン、インコーポレイテッド	異常ヘモグロビン症の治療のためのシステム及び方法
CN116349651A (zh)	2018-03-19	2023-06-30	瑞泽恩制药公司	使用crispr/cas系统对动物进行转录调制
BR112020021229A2 (pt)	2018-04-19	2021-02-02	The Regents Of The University Of California	composições e métodos para edição de genes
CN112020560B (zh) *	2018-04-25	2024-02-23	中国农业大学	一种RNA编辑的CRISPR/Cas效应蛋白及系统
EP3794130A4 (en)	2018-05-16	2022-07-27	Synthego Corporation	METHODS AND SYSTEMS FOR DESIGN AND USE OF GUIDE RNA
US11866719B1 (en)	2018-06-04	2024-01-09	Inari Agriculture Technology, Inc.	Heterologous integration of regulatory elements to alter gene expression in wheat cells and wheat plants
KR102035654B1 (ko) *	2018-07-23	2019-10-24	대한민국	항균 펩타이드를 생산하는 돌연변이 곤충 세포주 또는 이의 제조방법
US11479762B1 (en)	2018-08-31	2022-10-25	Inari Agriculture Technology, Inc.	Compositions, systems, and methods for genome editing
CN112955550A (zh) *	2018-09-12	2021-06-11	基础科学研究院	诱导具有突变基因的细胞的死亡的组合物以及通过使用所述组合物诱导具有突变基因的细胞的死亡的方法
EP3861120A4 (en)	2018-10-01	2023-08-16	North Carolina State University	RECOMBINANT TYPE I CRISPR-CAS SYSTEM
WO2020076976A1 (en)	2018-10-10	2020-04-16	Readcoor, Inc.	Three-dimensional spatial molecular indexing
KR102081962B1 (ko) *	2018-10-11	2020-02-26	충남대학교 산학협력단	아밀로이드 전구 단백질 유전자를 절단하기 위한 조성물
BR112021007289A2 (pt)	2018-10-18	2021-07-27	Intellia Therapeutics, Inc.	composições e métodos para tratar deficiência de alfa-1 antitripsina
CN114207130A (zh)	2018-10-18	2022-03-18	英特利亚治疗股份有限公司	用于从白蛋白基因座进行转基因表达的组合物和方法
SG11202103734PA (en)	2018-10-18	2021-05-28	Intellia Therapeutics Inc	Compositions and methods for expressing factor ix
JP2022512726A (ja)	2018-10-18	2022-02-07	インテリアセラピューティクス，インコーポレーテッド	核酸構築物及び使用方法
CN113136375B (zh) *	2018-10-29	2023-01-06	中国农业大学	新型CRISPR/Cas12f酶和系统
US20220010321A1 (en) *	2018-11-01	2022-01-13	Keygene N.V.	Dual guide rna for crispr/cas genome editing in plants cells
EP3650557B1 (en) *	2018-11-07	2021-10-20	Justus-Liebig-Universität Gießen	Method for determination of restriction efficiency of endonucleases mediating double-strand breaks in dna target sequences
US20210324420A1 (en) *	2018-11-16	2021-10-21	Osaka University	Method for producing genome-edited cell
KR20200071198A (ko)	2018-12-10	2020-06-19	네오이뮨텍, 인코퍼레이티드	Nrf2 발현 조절 기반 T 세포 항암면역치료법
EP3896155A4 (en) *	2018-12-12	2022-08-17	Kyushu University, National University Corporation	PRODUCTION PROCESS FOR GENOMEEDIT CELLS
US11166996B2 (en)	2018-12-12	2021-11-09	Flagship Pioneering Innovations V, Inc.	Anellovirus compositions and methods of use
KR20210105914A (ko)	2018-12-20	2021-08-27	리제너론 파마슈티칼스 인코포레이티드	뉴클레아제-매개 반복부 팽창
GB2616539B (en) *	2019-01-07	2023-12-20	Crisp Hr Therapeutics Inc	A non-toxic cas9 enzyme and application thereof
CN109868275A (zh) *	2019-03-12	2019-06-11	中国农业大学	CRISPR/Cas9介导的羊FGF5基因敲除和定点整合MTNR1A基因的方法
EP3769090B1 (en)	2019-03-18	2023-11-15	Regeneron Pharmaceuticals, Inc.	Crispr/cas dropout screening platform to reveal genetic vulnerabilities associated with tau aggregation
EP4317950A3 (en)	2019-03-18	2024-04-17	Regeneron Pharmaceuticals, Inc.	Crispr/cas screening platform to identify genetic modifiers of tau seeding or aggregation
WO2020191243A1 (en)	2019-03-19	2020-09-24	The Broad Institute, Inc.	Methods and compositions for editing nucleotide sequences
SG11202108451VA (en)	2019-04-03	2021-09-29	Regeneron Pharma	Methods and compositions for insertion of antibody coding sequences into a safe harbor locus
RU2771374C1 (ru)	2019-04-04	2022-05-04	Редженерон Фармасьютикалс, Инк.	Способы для бесшовного внесения целевых модификаций в направленные векторы
US11737435B2 (en)	2019-04-04	2023-08-29	Regeneron Pharmaceuticals, Inc.	Non-human animals comprising a humanized coagulation factor 12 locus
EA202192931A1 (ru)	2019-04-30	2022-02-22	Эдиджен Инк.	Способ прогнозирования эффективности лечения гемоглобинопатии
US11692197B2 (en)	2019-05-06	2023-07-04	Inari Agriculture Technology, Inc.	Delivery of biological molecules to plant cells
WO2020247452A1 (en)	2019-06-04	2020-12-10	Regeneron Pharmaceuticals, Inc.	Non-human animals comprising a humanized ttr locus with a beta-slip mutation and methods of use
CN111304180B (zh) *	2019-06-04	2023-05-26	山东舜丰生物科技有限公司	一种新的dna核酸切割酶及其应用
TW202112229A (zh)	2019-06-07	2021-04-01	美商雷傑納榮製藥公司	包含人化白蛋白基因座之非人的動物
CA3137765A1 (en)	2019-06-14	2020-12-17	Regeneron Pharmaceuticals, Inc.	Models of tauopathy
JP2022539338A (ja)	2019-06-25	2022-09-08	イナリアグリカルチャーテクノロジー，インコーポレイテッド	改良された相同性依存性修復ゲノム編集
US20220340969A1 (en)	2019-09-04	2022-10-27	Edigene Inc.	Method for evaluating gene editing therapy based on off-target assessment
JP2022548031A (ja)	2019-09-13	2022-11-16	リジェネロン・ファーマシューティカルズ・インコーポレイテッド	脂質ナノ粒子によって送達されるｃｒｉｓｐｒ／ｃａｓシステムを使用する動物における転写調節
WO2021061815A1 (en)	2019-09-23	2021-04-01	Omega Therapeutics, Inc.	COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
WO2021061707A1 (en)	2019-09-23	2021-04-01	Omega Therapeutics, Inc.	Compositions and methods for modulating apolipoprotein b (apob) gene expression
EP4038190A1 (en)	2019-10-03	2022-08-10	Artisan Development Labs, Inc.	Crispr systems with engineered dual guide nucleic acids
IL292434A (en) *	2019-11-05	2022-06-01	Pairwise Plants Services Inc	Preparations and methods for replacing DNA encoded by rna of alleles
US20230001019A1 (en)	2019-11-08	2023-01-05	Regeneron Pharmaceuticals, Inc.	Crispr and aav strategies for x-linked juvenile retinoschisis therapy
JP7448120B2 (ja) *	2019-11-14	2024-03-12	国立研究開発法人農業・食品産業技術総合研究機構	プラズマを用いてゲノム編集酵素を植物細胞内に導入する方法
WO2021108363A1 (en)	2019-11-25	2021-06-03	Regeneron Pharmaceuticals, Inc.	Crispr/cas-mediated upregulation of humanized ttr allele
WO2021108442A2 (en) *	2019-11-27	2021-06-03	The Regents Of The University Of California	Modulators of cas9 polypeptide activity and methods of use thereof
EP4086341A1 (en)	2019-12-30	2022-11-09	Edigene Biotechnology Inc.	Method for purifying ucart cell and use thereof
JP2023509058A (ja)	2019-12-30	2023-03-06	博雅▲緝▼因（北京）生物科技有限公司	Ｔ細胞リンパ腫細胞を標的とするユニバーサルｃａｒ－ｔならびにその調製方法及び使用
CN115052971A (zh) *	2020-01-14	2022-09-13	株式会社图尔金	在低氧条件下具有高适应性的细胞及其用途
KR102616080B1 (ko)	2020-01-14	2023-12-21	주식회사 툴젠	알츠하이머 예방 또는 치료용 약학적 조성물 및 이의 용도
WO2021163515A1 (en) *	2020-02-12	2021-08-19	Temple University - Of The Commonwealth System Of Higher Education	Crispr-cas9 mediated disruption of alcam gene inhibits adhesion and trans-endothelial migration of myeloid cells
KR20210109384A (ko)	2020-02-27	2021-09-06	주식회사 엘지화학	원형질체를 활용한 식물 유전자 교정 효율 증대 방법
CA3167369A1 (en)	2020-03-04	2021-09-10	Tara YOUNG	Methods and compositions for sensitization of tumor cells to immune therapy
WO2021183720A1 (en)	2020-03-11	2021-09-16	Omega Therapeutics, Inc.	Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
WO2021195079A1 (en)	2020-03-23	2021-09-30	Regeneron Pharmaceuticals, Inc.	Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use
WO2021202938A1 (en)	2020-04-03	2021-10-07	Creyon Bio, Inc.	Oligonucleotide-based machine learning
CA3177481A1 (en)	2020-05-08	2021-11-11	David R. Liu	Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN111690720B (zh) *	2020-06-16	2021-06-15	山东舜丰生物科技有限公司	利用修饰的单链核酸进行靶核酸检测的方法
US20240002839A1 (en)	2020-12-02	2024-01-04	Decibel Therapeutics, Inc.	Crispr sam biosensor cell lines and methods of use thereof
KR20220081949A (ko) *	2020-12-09	2022-06-16	재단법인 아산사회복지재단	온-타겟 활성이 유지되고 오프-타겟 활성이 감소된 가이드 rna 및 이의 용도
WO2022140560A1 (en)	2020-12-23	2022-06-30	Flagship Pioneering Innovations V, Inc.	In vitro assembly of anellovirus capsids enclosing rna
JP2024502036A (ja)	2020-12-30	2024-01-17	インテリアセラピューティクス，インコーポレイテッド	操作されたｔ細胞
KR102285906B1 (ko) *	2021-02-18	2021-08-04	주식회사 엔에이피	수생태계 교란어종 관리를 위한 큰입배스 불임 유도용 조성물
US20240247285A1 (en)	2021-05-10	2024-07-25	Sqz Biotechnologies Company	Methods for delivering genome editing molecules to the nucleus or cytosol of a cell and uses thereof
KR102616916B1 (ko) *	2021-05-11	2023-12-21	주식회사 애이마	발암성 돌연변이 유전자 검출용 가이드 rna 내지 이의 용도
WO2022251644A1 (en)	2021-05-28	2022-12-01	Lyell Immunopharma, Inc.	Nr4a3-deficient immune cells and uses thereof
WO2022256448A2 (en)	2021-06-01	2022-12-08	Artisan Development Labs, Inc.	Compositions and methods for targeting, editing, or modifying genes
US20230052243A1 (en)	2021-06-02	2023-02-16	Lyell Immunopharma, Inc.	Nr4a-deficient cells expressing c-jun and uses thereof
KR102618072B1 (ko)	2021-06-15	2023-12-28	대한민국	작물의 개화조절 관련 유전자 교정용 가이드 rna 및 이의 용도
WO2022266538A2 (en)	2021-06-18	2022-12-22	Artisan Development Labs, Inc.	Compositions and methods for targeting, editing or modifying human genes
EP4367242A2 (en)	2021-07-07	2024-05-15	Omega Therapeutics, Inc.	Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
JP2024527634A (ja)	2021-07-09	2024-07-25	ツールゲンインコーポレイテッド	酸化ストレス抵抗性を有する間葉系幹細胞、この製造方法および用途
KR20230019791A (ko)	2021-07-29	2023-02-09	주식회사 툴젠	혈액 적합성을 갖는 중간엽 줄기세포, 이의 제조방법 및 용도
CN113584134B (zh) *	2021-09-06	2024-01-30	青岛金斯达生物技术有限公司	一种基于CRISPR-Cas9的等温核酸检测系统及其方法和应用
AU2022343300A1 (en)	2021-09-10	2024-04-18	Agilent Technologies, Inc.	Guide rnas with chemical modification for prime editing
MX2024003887A (es)	2021-10-14	2024-07-09	Arsenal Biosciences Inc	Células inmunitarias que tienen arnch coespresados y sistemas de compuerta lógica.
EP4405463A1 (en)	2021-10-14	2024-07-31	Lonza Sales AG	Modified producer cells for extracellular vesicle production
EP4423271A2 (en)	2021-10-28	2024-09-04	Regeneron Pharmaceuticals, Inc.	Crispr/cas-related methods and compositions for knocking out c5
CA3237300A1 (en)	2021-11-01	2023-05-04	Tome Biosciences, Inc.	Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo
WO2023108047A1 (en)	2021-12-08	2023-06-15	Regeneron Pharmaceuticals, Inc.	Mutant myocilin disease model and uses thereof
EP4452335A1 (en)	2021-12-22	2024-10-30	Tome Biosciences, Inc.	Co-delivery of a gene editor construct and a donor template
AU2022417615A1 (en)	2021-12-23	2024-06-27	University Of Massachusetts	Therapeutic treatment for fragile x-associated disorder
KR20240128067A (ko)	2021-12-29	2024-08-23	브리스톨-마이어스 스큅 컴퍼니	랜딩 패드 세포주의 생성
WO2023150181A1 (en)	2022-02-01	2023-08-10	President And Fellows Of Harvard College	Methods and compositions for treating cancer
US20230338477A1 (en)	2022-02-02	2023-10-26	Regeneron Pharmaceuticals, Inc.	Anti-tfr:gaa and anti-cd63:gaa insertion for treatment of pompe disease
WO2023167882A1 (en)	2022-03-01	2023-09-07	Artisan Development Labs, Inc.	Composition and methods for transgene insertion
CN118843688A (zh)	2022-03-04	2024-10-25	株式会社图尔金	低免疫原性干细胞、由干细胞分化或衍生的低免疫原性细胞及其制备方法
CN114410609B (zh) *	2022-03-29	2022-07-12	舜丰生物科技(海南)有限公司	一种活性提高的Cas蛋白以及应用
WO2023205744A1 (en)	2022-04-20	2023-10-26	Tome Biosciences, Inc.	Programmable gene insertion compositions
WO2023205844A1 (en) *	2022-04-26	2023-11-02	Peter Maccallum Cancer Institute	Nucleic acids and uses thereof
WO2023212677A2 (en)	2022-04-29	2023-11-02	Regeneron Pharmaceuticals, Inc.	Identification of tissue-specific extragenic safe harbors for gene therapy approaches
WO2023215831A1 (en)	2022-05-04	2023-11-09	Tome Biosciences, Inc.	Guide rna compositions for programmable gene insertion
AU2023269134A1 (en)	2022-05-09	2024-12-12	Regeneron Pharmaceuticals, Inc.	Vectors and methods for in vivo antibody production
WO2023225665A1 (en)	2022-05-19	2023-11-23	Lyell Immunopharma, Inc.	Polynucleotides targeting nr4a3 and uses thereof
WO2023225670A2 (en)	2022-05-20	2023-11-23	Tome Biosciences, Inc.	Ex vivo programmable gene insertion
WO2023225410A2 (en)	2022-05-20	2023-11-23	Artisan Development Labs, Inc.	Systems and methods for assessing risk of genome editing events
WO2023230549A2 (en)	2022-05-25	2023-11-30	Flagship Pioneering Innovations Vii, Llc	Compositions and methods for modulation of tumor suppressors and oncogenes
AU2023277636A1 (en)	2022-05-25	2024-11-21	Flagship Pioneering Innovations Vii, Llc	Compositions and methods for modulating genetic drivers
WO2023230566A2 (en)	2022-05-25	2023-11-30	Flagship Pioneering Innovations Vii, Llc	Compositions and methods for modulating cytokines
WO2023230573A2 (en)	2022-05-25	2023-11-30	Flagship Pioneering Innovations Vii, Llc	Compositions and methods for modulation of immune responses
WO2023230578A2 (en)	2022-05-25	2023-11-30	Flagship Pioneering Innovations Vii, Llc	Compositions and methods for modulating circulating factors
WO2023235725A2 (en)	2022-05-31	2023-12-07	Regeneron Pharmaceuticals, Inc.	Crispr-based therapeutics for c9orf72 repeat expansion disease
WO2023235726A2 (en)	2022-05-31	2023-12-07	Regeneron Pharmaceuticals, Inc.	Crispr interference therapeutics for c9orf72 repeat expansion disease
WO2023250511A2 (en)	2022-06-24	2023-12-28	Tune Therapeutics, Inc.	Compositions, systems, and methods for reducing low-density lipoprotein through targeted gene repression
WO2024006955A1 (en)	2022-06-29	2024-01-04	Intellia Therapeutics, Inc.	Engineered t cells
EP4299733A1 (en)	2022-06-30	2024-01-03	Inari Agriculture Technology, Inc.	Compositions, systems, and methods for genome editing
WO2024005863A1 (en)	2022-06-30	2024-01-04	Inari Agriculture Technology, Inc.	Compositions, systems, and methods for genome editing
WO2024005864A1 (en)	2022-06-30	2024-01-04	Inari Agriculture Technology, Inc.	Compositions, systems, and methods for genome editing
EP4299739A1 (en)	2022-06-30	2024-01-03	Inari Agriculture Technology, Inc.	Compositions, systems, and methods for genome editing
WO2024020587A2 (en)	2022-07-22	2024-01-25	Tome Biosciences, Inc.	Pleiopluripotent stem cell programmable gene insertion
WO2024026474A1 (en)	2022-07-29	2024-02-01	Regeneron Pharmaceuticals, Inc.	Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
WO2024031053A1 (en)	2022-08-05	2024-02-08	Regeneron Pharmaceuticals, Inc.	Aggregation-resistant variants of tdp-43
CN115386597A (zh) *	2022-09-19	2022-11-25	国家纳米科学中心	一种提高基因载体转染效率和/或转染精度的转染辅助试剂及其应用
WO2024064958A1 (en)	2022-09-23	2024-03-28	Lyell Immunopharma, Inc.	Methods for culturing nr4a-deficient cells
WO2024064952A1 (en)	2022-09-23	2024-03-28	Lyell Immunopharma, Inc.	Methods for culturing nr4a-deficient cells overexpressing c-jun
WO2024073606A1 (en)	2022-09-28	2024-04-04	Regeneron Pharmaceuticals, Inc.	Antibody resistant modified receptors to enhance cell-based therapies
WO2024077174A1 (en)	2022-10-05	2024-04-11	Lyell Immunopharma, Inc.	Methods for culturing nr4a-deficient cells
WO2024098002A1 (en)	2022-11-04	2024-05-10	Regeneron Pharmaceuticals, Inc.	Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle
WO2024102434A1 (en)	2022-11-10	2024-05-16	Senda Biosciences, Inc.	Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs
US20240173426A1 (en)	2022-11-14	2024-05-30	Regeneron Pharmaceuticals, Inc.	Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes
WO2024138194A1 (en)	2022-12-22	2024-06-27	Tome Biosciences, Inc.	Platforms, compositions, and methods for in vivo programmable gene insertion
WO2024159071A1 (en)	2023-01-27	2024-08-02	Regeneron Pharmaceuticals, Inc.	Modified rhabdovirus glycoproteins and uses thereof
WO2024211287A1 (en)	2023-04-03	2024-10-10	Seagen Inc.	Production cell lines with targeted integration sites
WO2024218295A1 (en)	2023-04-21	2024-10-24	Vib Vzw	Allelic combinations in crops for yield increase

Citations (1)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US20140068797A1 (en) *	2012-05-25	2014-03-06	University Of Vienna	Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Family Cites Families (81)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
EP0262516A2 (en) *	1986-09-29	1988-04-06	Miles Inc.	Genetic transformation of lactic acid bacteria
US5350689A (en) *	1987-05-20	1994-09-27	Ciba-Geigy Corporation	Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells
IL90161A0 (en)	1988-05-13	1989-12-15	Phillips Petroleum Co	Expression of hepatitis b s and pres2 proteins in methylotrophic yeasts
US5767367A (en) *	1990-06-23	1998-06-16	Hoechst Aktiengesellschaft	Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation
US5925517A (en)	1993-11-12	1999-07-20	The Public Health Research Institute Of The City Of New York, Inc.	Detectably labeled dual conformation oligonucleotide probes, assays and kits
US6040295A (en) *	1995-01-13	2000-03-21	Genemedicine, Inc.	Formulated nucleic acid compositions and methods of administering the same for gene therapy
JPH1080274A (ja)	1996-09-06	1998-03-31	Hirofumi Hamada	核移行シグナルを結合したテトラサイクリン・トランスアクチベーター
FR2753204B1 (fr)	1996-09-11	1998-12-04	Transgene Sa	Procede de preparation d'adn plasmidique
EP1165842A4 (en)	1999-03-16	2004-07-07	Dana Farber Cancer Inst Inc	LENTIVIRAL VECTOR SYSTEMS FOR SCREENING LARGE QUANTITIES
AU2002254007B2 (en)	2001-02-22	2006-09-21	Purdue Research Foundation	Compositions based on vanilloid-catechin synergies for prevention and treatment of cancer
WO2002067996A2 (de)	2001-02-24	2002-09-06	Mologen Forschungs-, Entwicklungs- Und Vertriebs Gmbh	Beta-endorphin / crf - gentherapie zur lokalen schmerzbekämpfung
AU2011203213A1 (en)	2002-08-28	2011-08-04	Chromocell Corporation	Selection and Isolation of Living Cells Using mRNA-Binding Probes
JP2005006578A (ja) *	2003-06-20	2005-01-13	Nippon Genetech Co Ltd	イン・ビトロ転写反応を利用した機能性ｒｎａ分子の製造方法
SI1667678T1 (sl)	2003-10-03	2009-12-31	Veijlen N V	Sestavek za ĺ˝ivalsko hrano
AU2004294567A1 (en)	2003-11-26	2005-06-16	University Of Massachusetts	Sequence-specific inhibition of small RNA function
US20050220796A1 (en)	2004-03-31	2005-10-06	Dynan William S	Compositions and methods for modulating DNA repair
WO2005123962A2 (en) *	2004-06-14	2005-12-29	The University Of Texas At Austin	Gene targeting in eukaryotic cells by group ii intron ribonucleoprotein particles
US20070048741A1 (en)	2005-08-24	2007-03-01	Getts Robert C	Methods and kits for sense RNA synthesis
WO2007024029A1 (en)	2005-08-26	2007-03-01	Kyoto University	Antiviral agent and viral replication inhibitor
CN101688241B (zh)	2007-03-02	2015-01-21	杜邦营养生物科学有限公司	具有改善的噬菌体抗性的培养物
CN101878307B (zh) *	2007-09-27	2017-07-28	陶氏益农公司	以5‑烯醇式丙酮酰莽草酸‑3‑磷酸合酶基因为靶的改造锌指蛋白
US20100067918A1 (en)	2008-04-18	2010-03-18	New Jersey Institute Of Technology	Ultra-miniaturized thz communication device and system
WO2010001189A1 (en)	2008-07-03	2010-01-07	Cellectis	The crystal structure of i-dmoi in complex with its dna target, improved chimeric meganucleases and uses thereof
US20100076057A1 (en)	2008-09-23	2010-03-25	Northwestern University	TARGET DNA INTERFERENCE WITH crRNA
EP2184292A1 (en)	2008-11-10	2010-05-12	Ulrich Kunzendorf	Anti-Apoptotic Fusion Proteins
US8388824B2 (en)	2008-11-26	2013-03-05	Enthone Inc.	Method and composition for electrodeposition of copper in microelectronics with dipyridyl-based levelers
KR20100080068A (ko) *	2008-12-31	2010-07-08	주식회사 툴젠	신규한 징크 핑거 뉴클레아제 및 이의 용도
WO2011007193A1 (en)	2009-07-17	2011-01-20	Cellectis	Viral vectors encoding a dna repair matrix and containing a virion-associated site specific meganuclease for gene targeting
US8586526B2 (en)	2010-05-17	2013-11-19	Sangamo Biosciences, Inc.	DNA-binding proteins and uses thereof
US8846350B2 (en) *	2009-10-26	2014-09-30	Albert Einstein College Of Medicine Of Yeshiva University	MicroRNA affinity assay and uses thereof
US10087431B2 (en)	2010-03-10	2018-10-02	The Regents Of The University Of California	Methods of generating nucleic acid fragments
US9315825B2 (en)	2010-03-29	2016-04-19	The Trustees Of The University Of Pennsylvania	Pharmacologically induced transgene ablation system
WO2011130343A1 (en)	2010-04-13	2011-10-20	Sigma-Aldrich Co.	Biosensors comprising protein-binding domains and fluorescent proteins
BR112012028805A2 (pt) *	2010-05-10	2019-09-24	The Regents Of The Univ Of California E Nereus Pharmaceuticals Inc	composições de endorribonuclease e métodos de uso das mesmas.
JP2013537410A (ja)	2010-07-23	2013-10-03	シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニー	標的化エンドヌクレアーゼおよび一本鎖核酸を用いたゲノム編集
CN102358902B (zh) *	2011-04-02	2013-01-02	西南大学	家蚕丝素重链基因突变序列及突变的方法和应用
SG10201602651YA (en)	2011-04-05	2016-05-30	Cellectis	Method for the generation of compact tale-nucleases and uses thereof
GB201122458D0 (en)	2011-12-30	2012-02-08	Univ Wageningen	Modified cascade ribonucleoproteins and uses thereof
JP6170080B2 (ja)	2012-02-24	2017-07-26	フレッドハッチンソンキャンサーリサーチセンター	異常ヘモグロビン症の治療のための組成物および方法
US9637739B2 (en)	2012-03-20	2017-05-02	Vilnius University	RNA-directed DNA cleavage by the Cas9-crRNA complex
WO2013141680A1 (en) *	2012-03-20	2013-09-26	Vilnius University	RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
WO2013176776A1 (en)	2012-05-23	2013-11-28	Carrier Corporation	Wall panel for climate controlled cargo container
US20150128300A1 (en)	2012-06-12	2015-05-07	Genentech, Inc.	Methods and compositions for generating conditional knock-out alleles
CA2877290A1 (en)	2012-06-19	2013-12-27	Daniel F. Voytas	Gene targeting in plants using dna viruses
PL3494997T3 (pl) *	2012-07-25	2020-04-30	The Broad Institute, Inc.	Indukowalne białka wiążące dna i narzędzia perturbacji genomu oraz ich zastosowania
EP2880171B1 (en)	2012-08-03	2018-10-03	The Regents of The University of California	Methods and compositions for controlling gene expression by rna processing
US9963715B2 (en) *	2012-08-29	2018-05-08	Sangamo Therapeutics, Inc.	Methods and compositions for treatment of a genetic condition
NZ705745A (en)	2012-09-07	2018-11-30	Sangamo Biosciences Inc	Fad3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
UA118090C2 (uk) *	2012-09-07	2018-11-26	ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі	Спосіб інтегрування послідовності нуклеїнової кислоти, що представляє інтерес, у ген fad2 у клітині сої та специфічний для локусу fad2 білок, що зв'язується, здатний індукувати спрямований розрив
UA119135C2 (uk)	2012-09-07	2019-05-10	ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі	Спосіб отримання трансгенної рослини
WO2014065596A1 (en)	2012-10-23	2014-05-01	Toolgen Incorporated	Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
LT3138912T (lt)	2012-12-06	2019-02-25	Sigma-Aldrich Co. Llc	Genomo modifikavimas ir reguliavimas crispr pagrindu
EP2921557B1 (en)	2012-12-12	2016-07-13	The Broad Institute, Inc.	Engineering of systems, methods and optimized guide compositions for sequence manipulation
IL307735A (en)	2012-12-12	2023-12-01	Broad Inst Inc	Systems engineering, methods and optimal guiding components for sequence manipulation
CA2894684A1 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Engineering and optimization of improved crispr-cas systems, methods and enzyme compositions for sequence manipulation in eukaryotes
RU2721275C2 (ru)	2012-12-12	2020-05-18	Те Брод Инститьют, Инк.	Доставка, конструирование и оптимизация систем, способов и композиций для манипуляции с последовательностями и применения в терапии
WO2014093701A1 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
US8889559B2 (en)	2012-12-12	2014-11-18	Micron Technology, Inc.	Methods of forming a pattern on a substrate
US8697359B1 (en)	2012-12-12	2014-04-15	The Broad Institute, Inc.	CRISPR-Cas systems and methods for altering expression of gene products
WO2014093655A2 (en) *	2012-12-12	2014-06-19	The Broad Institute, Inc.	Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2014093694A1 (en)	2012-12-12	2014-06-19	The Broad Institute, Inc.	Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
JP2016505256A (ja)	2012-12-12	2016-02-25	ザ・ブロード・インスティテュート・インコーポレイテッ	配列操作のためのＣＲＩＳＰＲ−Ｃａｓ成分系、方法および組成物
EP2931892B1 (en)	2012-12-12	2018-09-12	The Broad Institute, Inc.	Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
IL239326B1 (en)	2012-12-17	2024-10-01	Harvard College	RNA-guided human genome engineering
ES2953523T3 (es) *	2012-12-27	2023-11-14	Keygene Nv	Método para inducir una translocación dirigida en una planta
CN103233028B (zh) *	2013-01-25	2015-05-13	南京徇齐生物技术有限公司	一种无物种限制无生物安全性问题的真核生物基因打靶方法及螺旋结构dna序列
JP2016519652A (ja) *	2013-03-14	2016-07-07	カリブー・バイオサイエンシーズ・インコーポレイテッド	核酸ターゲティング核酸の組成物および方法
CN105209624A (zh) *	2013-03-15	2015-12-30	明尼苏达大学董事会	采用CRISPR/Cas系统的植物基因组的工程改造
US20140273230A1 (en)	2013-03-15	2014-09-18	Sigma-Aldrich Co., Llc	Crispr-based genome modification and regulation
EP3778899A1 (en) *	2013-05-22	2021-02-17	Northwestern University	Rna-directed dna cleavage and gene editing by cas9 enzyme from neisseria meningitidis
US20150067922A1 (en) *	2013-05-30	2015-03-05	The Penn State Research Foundation	Gene targeting and genetic modification of plants via rna-guided genome editing
CA3176690A1 (en) *	2013-06-04	2014-12-11	President And Fellows Of Harvard College	Rna-guided transcriptional regulation
DK3011032T3 (da)	2013-06-17	2020-01-20	Broad Inst Inc	Fremføring, modificering og optimering af systemer, fremgangsmåder og sammensætninger til målretning mod og modellering af sygdomme og forstyrrelser i postmitotiske celler
DK3011031T3 (da)	2013-06-17	2020-12-21	Broad Inst Inc	Fremføring og anvendelse af crispr-cas-systemerne, vektorer og sammensætninger til levermålretning og -terapi
CN105492611A (zh) *	2013-06-17	2016-04-13	布罗德研究所有限公司	用于序列操纵的优化的crispr-cas双切口酶系统、方法以及组合物
WO2014204723A1 (en)	2013-06-17	2014-12-24	The Broad Institute Inc.	Oncogenic models based on delivery and use of the crispr-cas systems, vectors and compositions
JP6702858B2 (ja)	2013-06-17	2020-06-03	ザ・ブロード・インスティテュート・インコーポレイテッド	ウイルス成分を使用して障害および疾患をターゲティングするためのＣＲＩＳＰＲ−Ｃａｓ系および組成物の送達、使用および治療上の適用
EP3011029B1 (en)	2013-06-17	2019-12-11	The Broad Institute, Inc.	Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
CN103343120B (zh) *	2013-07-04	2015-03-04	中国科学院遗传与发育生物学研究所	一种小麦基因组定点改造方法
MX2016002306A (es) *	2013-08-22	2016-07-08	Du Pont	Promotor u6 de polimerasa iii de soja y metodos de uso.
WO2016036754A1 (en) *	2014-09-02	2016-03-10	The Regents Of The University Of California	Methods and compositions for rna-directed target dna modification

2013
- 2013-10-23 WO PCT/KR2013/009488 patent/WO2014065596A1/en active Application Filing
- 2013-10-23 EP EP24165204.9A patent/EP4397760A3/en active Pending
- 2013-10-23 EP EP23183112.4A patent/EP4342987A3/en active Pending
- 2013-10-23 CA CA2888190A patent/CA2888190C/en active Active
- 2013-10-23 EP EP18158147.1A patent/EP3372679A1/en active Pending
- 2013-10-23 BR BR112015008708A patent/BR112015008708A2/pt active Search and Examination
- 2013-10-23 KR KR1020157013868A patent/KR102182847B1/ko active IP Right Grant
- 2013-10-23 DE DE202013012597.7U patent/DE202013012597U1/de not_active Expired - Lifetime
- 2013-10-23 SG SG11201503059XA patent/SG11201503059XA/en unknown
- 2013-10-23 KR KR1020157022594A patent/KR101706085B1/ko active IP Right Grant
- 2013-10-23 CN CN201910950792.1A patent/CN110592089B/zh active Active
- 2013-10-23 EP EP13849670.8A patent/EP2912175B1/en not_active Revoked
- 2013-10-23 CA CA3233048A patent/CA3233048A1/en active Pending
- 2013-10-23 CN CN201380066348.4A patent/CN104968784B/zh active Active
- 2013-10-23 ES ES17206792T patent/ES2886448T3/es active Active
- 2013-10-23 CN CN201910137869.3A patent/CN110066775B/zh active Active
- 2013-10-23 US US14/438,098 patent/US20150284727A1/en not_active Abandoned
- 2013-10-23 DK DK13849670.8T patent/DK2912175T3/en active
- 2013-10-23 SG SG10201809566SA patent/SG10201809566SA/en unknown
- 2013-10-23 CN CN202211117242.XA patent/CN116064533A/zh active Pending
- 2013-10-23 PT PT13849670T patent/PT2912175T/pt unknown
- 2013-10-23 AU AU2013335451A patent/AU2013335451C1/en active Active
- 2013-10-23 CN CN201910950859.1A patent/CN110669746B/zh active Active
- 2013-10-23 KR KR1020157022592A patent/KR101656236B1/ko active IP Right Review Request
- 2013-10-23 CN CN201910950935.9A patent/CN110669758A/zh active Pending
- 2013-10-23 KR KR1020187006398A patent/KR102052286B1/ko active IP Right Grant
- 2013-10-23 CN CN202211116560.4A patent/CN116064532A/zh active Pending
- 2013-10-23 JP JP2015538033A patent/JP6517143B2/ja active Active
- 2013-10-23 EP EP17206792.8A patent/EP3346003B1/en active Active
- 2013-10-23 DE DE202013012610.8U patent/DE202013012610U1/de not_active Expired - Lifetime
- 2013-10-23 CN CN201910950683.XA patent/CN110643600A/zh active Pending
- 2013-10-23 EP EP24165188.4A patent/EP4397759A3/en active Pending
- 2013-10-23 ES ES13849670T patent/ES2690386T3/es active Active
- 2013-10-23 PL PL13849670T patent/PL2912175T3/pl unknown
- 2013-10-23 ES ES20164644T patent/ES2926021T3/es active Active
- 2013-10-23 EP EP24158339.2A patent/EP4357457B1/en active Active
- 2013-10-23 EP EP20164644.5A patent/EP3733847B1/en active Active
- 2013-10-23 EP EP22176546.4A patent/EP4180526A3/en active Pending
- 2013-10-23 CN CN201510884156.5A patent/CN105441440B/zh active Active
- 2013-10-23 SG SG10201809564YA patent/SG10201809564YA/en unknown
- 2013-10-23 KR KR1020157022593A patent/KR101656237B1/ko active IP Right Review Request
2015
- 2015-04-13 US US14/685,568 patent/US10851380B2/en active Active
- 2015-04-13 US US14/685,510 patent/US20150344912A1/en active Pending
- 2015-08-27 AU AU2015218519A patent/AU2015218519A1/en not_active Abandoned
- 2015-09-08 JP JP2015176407A patent/JP2016027807A/ja active Pending
2016
- 2016-01-19 HK HK16100558.6A patent/HK1212732A1/zh unknown
- 2016-09-27 HK HK16111313.9A patent/HK1223125A1/zh unknown
2017
- 2017-08-10 JP JP2017155410A patent/JP2018019698A/ja active Pending
- 2017-10-31 AU AU2017254854A patent/AU2017254854B2/en active Active
2018
- 2018-12-21 HK HK18116451.8A patent/HK1257302A1/zh unknown
- 2018-12-21 HK HK18116450.9A patent/HK1257301A1/zh unknown
2020
- 2020-01-06 JP JP2020000091A patent/JP2020078303A/ja active Pending
- 2020-02-28 AU AU2020201485A patent/AU2020201485A1/en not_active Abandoned
- 2020-08-27 US US17/004,355 patent/US20210047648A1/en active Pending
- 2020-08-27 US US17/004,338 patent/US20210047647A1/en active Pending
2022
- 2022-03-16 JP JP2022041576A patent/JP2022095663A/ja active Pending
- 2022-07-07 AU AU2022204902A patent/AU2022204902A1/en active Pending
2023
- 2023-05-08 US US18/314,050 patent/US20230374525A1/en active Pending
- 2023-05-08 US US18/313,946 patent/US20230374524A1/en active Pending
- 2023-08-10 JP JP2023131350A patent/JP2023166401A/ja active Pending
- 2023-08-10 JP JP2023131349A patent/JP7571226B2/ja active Active
- 2023-09-15 US US18/467,952 patent/US20240240192A9/en active Pending
- 2023-09-15 US US18/467,967 patent/US20240026367A1/en active Pending
2024
- 2024-04-22 JP JP2024068959A patent/JP2024099674A/ja active Pending

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US20140068797A1 (en) *	2012-05-25	2014-03-06	University Of Vienna	Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Chiu et al in Engineered GFP as a viral reporter in plants (Curr. Biol. Vol 6, No 3, pages 325-330). *
Jinek et al A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity (Science Vol 337, published online June 28, 2012). *
Perez-Rodriquez (Dissertation Abstract first available to public on June 17, 2010). *
Perez-Rodriquez (Dissertation, first available to public on June 17, 2010). *

Cited By (153)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US10844378B2 (en)	2012-03-20	2020-11-24	Vilnius University	RNA-directed DNA cleavage by the Cas9-crRNA complex
US11555187B2 (en)	2012-03-20	2023-01-17	Vilnius University	RNA-directed DNA cleavage by the Cas9-crRNA complex
US20150050699A1 (en) *	2012-03-20	2015-02-19	Vilnius University	RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US10900054B2 (en)	2012-05-25	2021-01-26	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10400253B2 (en)	2012-05-25	2019-09-03	The Regents Of The University Of California	Methods and compositions or RNA-directed target DNA modification and for RNA-directed modulation of transcription
US12123015B2 (en)	2012-05-25	2024-10-22	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11970711B2 (en)	2012-05-25	2024-04-30	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11814645B2 (en)	2012-05-25	2023-11-14	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11674159B2 (en)	2012-05-25	2023-06-13	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11634730B2 (en)	2012-05-25	2023-04-25	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11549127B2 (en)	2012-05-25	2023-01-10	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11479794B2 (en)	2012-05-25	2022-10-25	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11473108B2 (en)	2012-05-25	2022-10-18	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11401532B2 (en)	2012-05-25	2022-08-02	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10000772B2 (en)	2012-05-25	2018-06-19	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11332761B2 (en)	2012-05-25	2022-05-17	The Regenis of Wie University of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11293034B2 (en)	2012-05-25	2022-04-05	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10113167B2 (en)	2012-05-25	2018-10-30	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11274318B2 (en)	2012-05-25	2022-03-15	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11242543B2 (en)	2012-05-25	2022-02-08	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11186849B2 (en)	2012-05-25	2021-11-30	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11028412B2 (en)	2012-05-25	2021-06-08	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008589B2 (en)	2012-05-25	2021-05-18	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10227611B2 (en)	2012-05-25	2019-03-12	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008590B2 (en)	2012-05-25	2021-05-18	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11001863B2 (en)	2012-05-25	2021-05-11	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10266850B2 (en)	2012-05-25	2019-04-23	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988782B2 (en)	2012-05-25	2021-04-27	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988780B2 (en)	2012-05-25	2021-04-27	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982230B2 (en)	2012-05-25	2021-04-20	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10301651B2 (en)	2012-05-25	2019-05-28	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10308961B2 (en)	2012-05-25	2019-06-04	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982231B2 (en)	2012-05-25	2021-04-20	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10337029B2 (en)	2012-05-25	2019-07-02	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10351878B2 (en)	2012-05-25	2019-07-16	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358658B2 (en)	2012-05-25	2019-07-23	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358659B2 (en)	2012-05-25	2019-07-23	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10385360B2 (en)	2012-05-25	2019-08-20	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10612045B2 (en)	2012-05-25	2020-04-07	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10793878B1 (en)	2012-05-25	2020-10-06	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10407697B2 (en)	2012-05-25	2019-09-10	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10415061B2 (en)	2012-05-25	2019-09-17	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10421980B2 (en)	2012-05-25	2019-09-24	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10774344B1 (en)	2012-05-25	2020-09-15	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10428352B2 (en)	2012-05-25	2019-10-01	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10752920B2 (en)	2012-05-25	2020-08-25	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10443076B2 (en)	2012-05-25	2019-10-15	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10676759B2 (en)	2012-05-25	2020-06-09	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10669560B2 (en)	2012-05-25	2020-06-02	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10487341B2 (en)	2012-05-25	2019-11-26	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10640791B2 (en)	2012-05-25	2020-05-05	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10513712B2 (en)	2012-05-25	2019-12-24	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10626419B2 (en)	2012-05-25	2020-04-21	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10519467B2 (en)	2012-05-25	2019-12-31	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10526619B2 (en)	2012-05-25	2020-01-07	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10533190B2 (en)	2012-05-25	2020-01-14	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10550407B2 (en)	2012-05-25	2020-02-04	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10563227B2 (en)	2012-05-25	2020-02-18	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10570419B2 (en)	2012-05-25	2020-02-25	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10577631B2 (en)	2012-05-25	2020-03-03	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10597680B2 (en)	2012-05-25	2020-03-24	The Regents Of The University Of California	Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10731181B2 (en)	2012-12-06	2020-08-04	Sigma, Aldrich Co. LLC	CRISPR-based genome modification and regulation
US10745716B2 (en)	2012-12-06	2020-08-18	Sigma-Aldrich Co. Llc	CRISPR-based genome modification and regulation
US12018272B2 (en)	2012-12-17	2024-06-25	President And Fellows Of Harvard College	RNA-guided human genome engineering
US20160002670A1 (en) *	2012-12-17	2016-01-07	President And Fellows Of Harvard College	RNA-Guided Human Genome Engineering
US9970024B2 (en)	2012-12-17	2018-05-15	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11512325B2 (en)	2012-12-17	2022-11-29	President And Fellows Of Harvard College	RNA-guided human genome engineering
US10273501B2 (en) *	2012-12-17	2019-04-30	President And Fellows Of Harvard College	RNA-guided human genome engineering
US10717990B2 (en)	2012-12-17	2020-07-21	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11359211B2 (en)	2012-12-17	2022-06-14	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11365429B2 (en)	2012-12-17	2022-06-21	President And Fellows Of Harvard College	RNA-guided human genome engineering
US10435708B2 (en)	2012-12-17	2019-10-08	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11535863B2 (en)	2012-12-17	2022-12-27	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11236359B2 (en)	2012-12-17	2022-02-01	President And Fellows Of Harvard College	RNA-guided human genome engineering
US11312953B2 (en)	2013-03-14	2022-04-26	Caribou Biosciences, Inc.	Compositions and methods of nucleic acid-targeting nucleic acids
US20160186208A1 (en) *	2013-04-16	2016-06-30	Whitehead Institute For Biomedical Research	Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
US11459585B2 (en) *	2013-07-09	2022-10-04	President And Fellows Of Harvard College	Multiplex RNA-guided genome engineering
US20160168592A1 (en) *	2013-07-09	2016-06-16	President And Fellows Of Harvard College	Multiplex RNA-Guided Genome Engineering
US10519457B2 (en)	2013-08-22	2019-12-31	E I Du Pont De Nemours And Company	Soybean U6 polymerase III promoter and methods of use
US11773400B2 (en)	2013-08-22	2023-10-03	E.I. Du Pont De Nemours And Company	Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof
US11390887B2 (en)	2013-11-07	2022-07-19	Editas Medicine, Inc.	CRISPR-related methods and compositions with governing gRNAS
US10640788B2 (en)	2013-11-07	2020-05-05	Editas Medicine, Inc.	CRISPR-related methods and compositions with governing gRNAs
US10190137B2 (en)	2013-11-07	2019-01-29	Editas Medicine, Inc.	CRISPR-related methods and compositions with governing gRNAS
US20210147879A1 (en) *	2013-11-19	2021-05-20	President And Fellows Of Harvard College	Large Gene Excision and Insertion
US10286084B2 (en)	2014-02-18	2019-05-14	Duke University	Compositions for the inactivation of virus replication and methods of making and using the same
US20150376587A1 (en) *	2014-06-25	2015-12-31	Caribou Biosciences, Inc.	RNA Modification to Engineer Cas9 Activity
US11680268B2 (en)	2014-11-07	2023-06-20	Editas Medicine, Inc.	Methods for improving CRISPR/Cas-mediated genome-editing
US10993419B2 (en)	2014-12-10	2021-05-04	Regents Of The University Of Minnesota	Genetically modified cells, tissues, and organs for treating disease
US10278372B2 (en)	2014-12-10	2019-05-07	Regents Of The University Of Minnesota	Genetically modified cells, tissues, and organs for treating disease
US9888673B2 (en)	2014-12-10	2018-02-13	Regents Of The University Of Minnesota	Genetically modified cells, tissues, and organs for treating disease
US11234418B2 (en)	2014-12-10	2022-02-01	Regents Of The University Of Minnesota	Genetically modified cells, tissues, and organs for treating disease
US10450576B2 (en)	2015-03-27	2019-10-22	E I Du Pont De Nemours And Company	Soybean U6 small nuclear RNA gene promoters and their use in constitutive expression of small RNA genes in plants
US11390884B2 (en)	2015-05-11	2022-07-19	Editas Medicine, Inc.	Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11555208B2 (en)	2015-06-03	2023-01-17	Board Of Regents Of The University Of Nebraska	DNA editing using relatively long single-stranded DNA and CRISPR/Cas9 to increase success rate in methods for preparing transgenic embryos and animals
US11549126B2 (en)	2015-06-03	2023-01-10	Board Of Regents Of The University Of Nebraska	Treatment methods using DNA editing with single-stranded DNA
US11911415B2 (en)	2015-06-09	2024-02-27	Editas Medicine, Inc.	CRISPR/Cas-related methods and compositions for improving transplantation
US11414657B2 (en)	2015-06-29	2022-08-16	Ionis Pharmaceuticals, Inc.	Modified CRISPR RNA and modified single CRISPR RNA and uses thereof
US11642375B2 (en)	2015-07-31	2023-05-09	Intima Bioscience, Inc.	Intracellular genomic transplant and methods of therapy
US11147837B2 (en)	2015-07-31	2021-10-19	Regents Of The University Of Minnesota	Modified cells and methods of therapy
US11642374B2 (en)	2015-07-31	2023-05-09	Intima Bioscience, Inc.	Intracellular genomic transplant and methods of therapy
US10166255B2 (en)	2015-07-31	2019-01-01	Regents Of The University Of Minnesota	Intracellular genomic transplant and methods of therapy
US10406177B2 (en)	2015-07-31	2019-09-10	Regents Of The University Of Minnesota	Modified cells and methods of therapy
US11903966B2 (en)	2015-07-31	2024-02-20	Regents Of The University Of Minnesota	Intracellular genomic transplant and methods of therapy
US11266692B2 (en)	2015-07-31	2022-03-08	Regents Of The University Of Minnesota	Intracellular genomic transplant and methods of therapy
US11925664B2 (en)	2015-07-31	2024-03-12	Intima Bioscience, Inc.	Intracellular genomic transplant and methods of therapy
US11583556B2 (en)	2015-07-31	2023-02-21	Regents Of The University Of Minnesota	Modified cells and methods of therapy
EP3461894A1 (en)	2015-08-07	2019-04-03	Caribou Biosciences, Inc.	Engineered crispr-cas9 compositions and methods of use
US11667911B2 (en)	2015-09-24	2023-06-06	Editas Medicine, Inc.	Use of exonucleases to improve CRISPR/CAS-mediated genome editing
US11149281B2 (en)	2015-10-06	2021-10-19	Institute For Basic Science	Method for producing genome-modified plants from plant protoplasts at high efficiency
US10138472B2 (en)	2015-10-23	2018-11-27	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US9745562B2 (en)	2015-10-23	2017-08-29	Caribou Biosciences, Inc.	Methods of using engineered nucleic-acid targeting nucleic acids
US10501728B2 (en)	2015-10-23	2019-12-10	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US10711258B2 (en)	2015-10-23	2020-07-14	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US10196619B1 (en)	2015-10-23	2019-02-05	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US9816081B1 (en)	2015-10-23	2017-11-14	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US10125354B1 (en)	2015-10-23	2018-11-13	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US10023853B1 (en)	2015-10-23	2018-07-17	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US9957490B1 (en)	2015-10-23	2018-05-01	Caribou Biosciences, Inc.	Cells comprising engineered nucleic-acid targeting nucleic acids
US9677090B2 (en)	2015-10-23	2017-06-13	Caribou Biosciences, Inc.	Engineered nucleic-acid targeting nucleic acids
US9771600B2 (en)	2015-12-04	2017-09-26	Caribou Biosciences, Inc.	Engineered nucleic acid-targeting nucleic acids
US11505808B2 (en)	2015-12-04	2022-11-22	Caribou Biosciences, Inc.	Engineered nucleic acid-targeting nucleic acids
US9970029B1 (en)	2015-12-04	2018-05-15	Caribou Biosciences, Inc.	Engineered nucleic acid-targeting nucleic acids
US10100333B2 (en)	2015-12-04	2018-10-16	Caribou Biosciences, Inc.	Engineered nucleic acid-targeting nucleic acids
US11773411B2 (en)	2016-01-11	2023-10-03	The Board Of Trustees Of The Leland Stanford Junior University	Chimeric proteins and methods of regulating gene expression
US10457961B2 (en)	2016-01-11	2019-10-29	The Board Of Trustees Of The Leland Stanford Junior University	Chimeric proteins and methods of regulating gene expression
US11111287B2 (en)	2016-01-11	2021-09-07	The Board Of Trustees Of The Leland Stanford Junior University	Chimeric proteins and methods of immunotherapy
US9856497B2 (en)	2016-01-11	2018-01-02	The Board Of Trustee Of The Leland Stanford Junior University	Chimeric proteins and methods of regulating gene expression
US10336807B2 (en)	2016-01-11	2019-07-02	The Board Of Trustees Of The Leland Stanford Junior University	Chimeric proteins and methods of immunotherapy
US11597924B2 (en)	2016-03-25	2023-03-07	Editas Medicine, Inc.	Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US12049651B2 (en)	2016-04-13	2024-07-30	Editas Medicine, Inc.	Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11236313B2 (en)	2016-04-13	2022-02-01	Editas Medicine, Inc.	Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11912987B2 (en)	2016-08-03	2024-02-27	KSQ Therapeutics, Inc.	Methods for screening for cancer targets
US11078481B1 (en)	2016-08-03	2021-08-03	KSQ Therapeutics, Inc.	Methods for screening for cancer targets
US11078483B1 (en)	2016-09-02	2021-08-03	KSQ Therapeutics, Inc.	Methods for measuring and improving CRISPR reagent function
US11946163B2 (en)	2016-09-02	2024-04-02	KSQ Therapeutics, Inc.	Methods for measuring and improving CRISPR reagent function
US11154574B2 (en)	2016-10-18	2021-10-26	Regents Of The University Of Minnesota	Tumor infiltrating lymphocytes and methods of therapy
US10912797B2 (en)	2016-10-18	2021-02-09	Intima Bioscience, Inc.	Tumor infiltrating lymphocytes and methods of therapy
US12110545B2 (en)	2017-01-06	2024-10-08	Editas Medicine, Inc.	Methods of assessing nuclease cleavage
US11466271B2 (en)	2017-02-06	2022-10-11	Novartis Ag	Compositions and methods for the treatment of hemoglobinopathies
US12058986B2 (en)	2017-04-20	2024-08-13	Egenesis, Inc.	Method for generating a genetically modified pig with inactivated porcine endogenous retrovirus (PERV) elements
US11499151B2 (en)	2017-04-28	2022-11-15	Editas Medicine, Inc.	Methods and systems for analyzing guide RNA molecules
US11098297B2 (en)	2017-06-09	2021-08-24	Editas Medicine, Inc.	Engineered Cas9 nucleases
US10428319B2 (en)	2017-06-09	2019-10-01	Editas Medicine, Inc.	Engineered Cas9 nucleases
US11098325B2 (en)	2017-06-30	2021-08-24	Intima Bioscience, Inc.	Adeno-associated viral vectors for gene therapy
US11866726B2 (en)	2017-07-14	2024-01-09	Editas Medicine, Inc.	Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US20200158716A1 (en) *	2017-07-17	2020-05-21	Massachusetts Institute Of Technology	Cell atlas of healthy and diseased barrier tissues
US11713452B2 (en)	2017-09-08	2023-08-01	University Of North Texas Health Science Center	Engineered CAS9 variants
WO2019051419A1 (en) *	2017-09-08	2019-03-14	University Of North Texas Health Science Center	MODIFIED CASE VARIANTS9
US11572574B2 (en)	2017-09-28	2023-02-07	Toolgen Incorporated	Artificial genome manipulation for gene expression regulation
US12084676B2 (en)	2018-02-23	2024-09-10	Pioneer Hi-Bred International, Inc.	Cas9 orthologs
US11807878B2 (en)	2018-12-14	2023-11-07	Pioneer Hi-Bred International, Inc.	CRISPR-Cas systems for genome editing
US10934536B2 (en)	2018-12-14	2021-03-02	Pioneer Hi-Bred International, Inc.	CRISPR-CAS systems for genome editing
US20210054371A1 (en) *	2019-08-19	2021-02-25	Minghong Zhong	Conjugates of Guide RNA-Cas Protein Complex

Also Published As

Publication number	Publication date
EP2912175B1 (en)	2018-08-22
US20150322457A1 (en)	2015-11-12
HK1257301A1 (zh)	2019-10-18
CN104968784B (zh)	2019-11-05
KR20150101476A (ko)	2015-09-03
US20240052356A1 (en)	2024-02-15
CN110066775A (zh)	2019-07-30
EP3346003A1 (en)	2018-07-11
EP4397759A2 (en)	2024-07-10
EP4397760A3 (en)	2024-10-09
US20230374524A1 (en)	2023-11-23
CA2888190C (en)	2024-04-30
SG10201809566SA (en)	2018-11-29
EP4357457B1 (en)	2024-10-16
CN110592089B (zh)	2022-11-01
ES2926021T3 (es)	2022-10-21
WO2014065596A1 (en)	2014-05-01
AU2015218519A1 (en)	2015-09-17
JP2020078303A (ja)	2020-05-28
DK2912175T3 (en)	2018-10-22
EP2912175A1 (en)	2015-09-02
EP3733847B1 (en)	2022-06-01
CN110592089A (zh)	2019-12-20
HK1223125A1 (zh)	2017-07-21
HK1257302A1 (zh)	2019-10-18
HK1212732A1 (zh)	2016-06-17
AU2013335451C1 (en)	2024-07-04
CA2888190A1 (en)	2014-05-01
CN104968784A (zh)	2015-10-07
US20240026367A1 (en)	2024-01-25
KR20150101477A (ko)	2015-09-03
JP6517143B2 (ja)	2019-05-22
EP4342987A3 (en)	2024-06-26
AU2013335451A1 (en)	2015-05-07
AU2020201485A1 (en)	2020-03-19
CN110643600A (zh)	2020-01-03
KR102182847B1 (ko)	2020-11-27
JP2016500003A (ja)	2016-01-07
EP4180526A3 (en)	2023-06-14
US10851380B2 (en)	2020-12-01
CN105441440A (zh)	2016-03-30
EP4397760A2 (en)	2024-07-10
EP4357457A3 (en)	2024-07-17
EP3346003B1 (en)	2021-06-09
EP4180526A2 (en)	2023-05-17
US20210047647A1 (en)	2021-02-18
PL2912175T3 (pl)	2019-03-29
CN110669746B (zh)	2024-04-16
EP3372679A1 (en)	2018-09-12
BR112015008708A2 (pt)	2017-09-26
DE202013012610U1 (de)	2017-11-24
US20210047648A1 (en)	2021-02-18
EP4357457A2 (en)	2024-04-24
ES2690386T3 (es)	2018-11-20
CN110669746A (zh)	2020-01-10
AU2017254854A1 (en)	2017-11-16
EP2912175A4 (en)	2015-12-09
CA3233048A1 (en)	2014-05-01
CN105441440B (zh)	2020-12-15
US20230374525A1 (en)	2023-11-23
PT2912175T (pt)	2018-11-05
SG11201503059XA (en)	2015-06-29
EP4397759A3 (en)	2024-10-09
JP2023166400A (ja)	2023-11-21
CN110669758A (zh)	2020-01-10
AU2022204902A1 (en)	2022-08-18
CN116064532A (zh)	2023-05-05
JP2016027807A (ja)	2016-02-25
JP2022095663A (ja)	2022-06-28
KR20180029092A (ko)	2018-03-19
US20240240192A9 (en)	2024-07-18
AU2017254854B2 (en)	2019-12-05
ES2886448T3 (es)	2021-12-20
CN110066775B (zh)	2024-03-19
US20150344912A1 (en)	2015-12-03
DE202013012597U1 (de)	2017-11-21
SG10201809564YA (en)	2018-11-29
JP7571226B2 (ja)	2024-10-22
KR20150101478A (ko)	2015-09-03
KR20150101446A (ko)	2015-09-03
KR101706085B1 (ko)	2017-02-14
KR101656236B1 (ko)	2016-09-12
JP2024099674A (ja)	2024-07-25
AU2013335451B2 (en)	2016-09-15
JP2023166401A (ja)	2023-11-21
JP2018019698A (ja)	2018-02-08
EP3733847A1 (en)	2020-11-04
EP4342987A2 (en)	2024-03-27
KR102052286B1 (ko)	2019-12-06
CN116064533A (zh)	2023-05-05
KR101656237B1 (ko)	2016-09-12

Legal Events

Date

Code

Title

Description

2015-05-28

AS

Assignment

Owner name: TOOLGEN INCORPORATED, KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIM, JIN-SOO;CHO, SEUNG WOO;KIM, SOJUNG;AND OTHERS;SIGNING DATES FROM 20150515 TO 20150520;REEL/FRAME:035733/0913

2016-10-10

STCB

Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION