EP4087923A1 - Cell populations with rationally designed edits - Google Patents
Cell populations with rationally designed editsInfo
- Publication number
- EP4087923A1 EP4087923A1 EP21738541.8A EP21738541A EP4087923A1 EP 4087923 A1 EP4087923 A1 EP 4087923A1 EP 21738541 A EP21738541 A EP 21738541A EP 4087923 A1 EP4087923 A1 EP 4087923A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- editing
- cells
- module
- cell
- nickase
- Prior art date
- 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.)
- Withdrawn
Links
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- 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
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- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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- 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
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Definitions
- the present disclosure relates to methods and compositions to increase the percentage of edited mammalian cells in a cell population when using nucleic-acid guided editing, as well as automated multi-module instruments for performing these methods using the disclosed compositions.
- nucleases have been identified that allow for manipulation of gene sequences, and hence gene function.
- the nucleases include nucleic acid-guided nucleases, which enable researchers to generate permanent edits in live cells.
- the present disclosure relates to methods, compositions, modules and automated multi-module cell processing instruments that increase the efficiency of nucleic-acid guided editing in a cell population, e.g., a mammalian cell population.
- methods presented herein include methods for increasing the rate of targeted editing using non-homologous end joining (NHEJ) repair, base editing, microhomology-directed repair (MMEJ) and/or homology-directed repair (HDR).
- NHEJ non-homologous end joining
- MMEJ microhomology-directed repair
- HDR homology-directed repair
- the disclosure provides methods for improving nuclease- directed editing of cells using enrichment means to identify cells that have received the editing components needed to perform the intended editing operation. Enrichment can be performed directly or using surrogates, e.g., cell surface handles co-introduced with one or more components of the editing components.
- the disclosure provides methods for improving nuclease- directed editing of cells using enrichment means to identify cells that have received the editing components needed to perform the intended editing operation.
- the enrichment handle and method can be based on a positive versus negative signal of the surrogate.
- the enrichment method can be based on a threshold level of a surrogate, e.g., a high level of an enrichment handle versus a low or absent level of an enrichment handle.
- the disclosure provides methods for improving nuclease- directed editing rates by enriching for mammalian cells that have received an HDR donor, e.g., identifying cells that are more likely to have received the editing apparatus along with the designs encoding the enrichment handle.
- the disclosure provides methods for improving nuclease- directed editing of mammalian cells using enrichment means to identifying mammalian cells that have received the HDR donor, the guide nucleic acid, and/or the nuclease.
- enrichment may involve a single enrichment method for HDR donor, the guide nucleic acid, and the nuclease, or two or more separate enrichment events for one or more of these elements.
- the HDR donor and guide nucleic acid may be introduced separately or covalently linked, as disclosed in, e.g., USPN 9,982,278.
- the disclosure provides methods of enriching for the editing efficiency of a target region in a cell population, the method comprising contacting a population of two or more cells with editing machinery comprising (a) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeting a first target region, wherein the gRNA is covalently attached to a region homologous to said first target region comprising an intended change in sequence relative to said target region, (b) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeting a second target region, wherein the gRNA is covalently attached to a region encoding a selectable marker and (c) a nuclease compatible with said gRNA sequence, exposing the population of cells to conditions to allow the cells to edit at the first and second target regions; and enriching for the cells from the population that express the selectable marker, wherein the selectable marker serves as a surrogate for editing of the first target region in the enriched
- the disclosure provides a method of increasing the editing efficiency of a cell population, the method comprising contacting a population of two or more cells with editing machinery comprising (a) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeting a first target region, wherein the gRNA is covalently attached to a region homologous to said first target region comprising an intended change in sequence relative to said target region, (b) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeting a second target region, wherein the gRNA is covalently attached to a region encoding a selectable marker, and (c) nucleic acids encoding a nuclease compatible with said gRNA sequence, exposing the population of cells to conditions to allow the cells to edit at the first and second target regions, and enriching for the cells from the population that express the selectable marker, wherein the selectable marker serves as a surrogate for editing of the first target region in
- the cell enrichment uses a physical enrichment of the cells expressing the selectable marker. Examples of this include fluorescent-activated cell sorting selection, magnetic-activated cell sorting selection, antibiotic selection, and the like.
- the cell enrichment uses a computational enrichment based on the presence of a selectable marker.
- the editing cassette targeting the first target region further comprises a barcode.
- the method further comprises incorporation of site-specific genomic barcodes that enable tracking of individual edited cells within a population.
- the HDR is improved using fusion proteins that retain certain characteristics of RNA-directed nucleases (e.g., the binding specificity and ability to cleave one or more DNA strands) and also utilize other enzymatic activities, e.g., replication inhibition, reverse transcriptase activity, transcription enhancement activity, and the like.
- These nuclease fusion proteins can be used in nuclease-directed editing using the disclosed methods, with or without the enrichment methods as disclosed herein.
- the HDR donor and guide nucleic acid may be introduced separately or covalently linked, as disclosed in, e.g., USPN 9,982,278.
- the HDR is improved using fusion proteins that retain the binding function and nickase activity of an RNA-directed nuclease and also utilize other enzymatic activities, e.g., replication inhibition, reverse transcriptase activity, transcription enhancement activity, and the like.
- nickase fusion proteins can be used in RNA-directed nickase editing using the disclosed methods, with or without the enrichment methods as disclosed herein.
- the HDR donor and guide nucleic acid may be introduced separately or covalently linked, as disclosed in, e.g., USPN 9,982,278.
- nickase can be introduced using DNA coding for the nickase introduced separately or covalently linked to the donor and guide DNA, or introduced separately in protein form.
- the editing methods include the use of a fusion protein with nucleic acids having a guide RNA covalently attached to a region homologous to a target region that contains one or more changes from the native target sequence, and preferably at least one enrichment mechanism, physical or computational.
- Such methods can use a single guide RNA construct, or use two or more guide RNA constructs to target different genomic locations.
- the nucleic acids contain multiple guide RNAs covalently attached to different target regions within the genome.
- the editing methods include the use of a nickase fusion protein with nucleic acids having a guide RNA covalently attached to a region homologous to a target region that contains one or more changes from the native target sequence, and at least one enrichment mechanism, physical or computational.
- the cells receiving the HDR donor can be enriched using an initial enrichment step, e.g., using an antibiotic selection or fluorescent detection, following by an enrichment step using an enrichment of the cells receiving and expressing the co-introduced cell surface antigen.
- Numerous enrichment handles may be used in the methods and instruments of the disclosure, including but not limited to various cell surface molecules linked to tag, e.g., a hemagglutinin (HA) tag, a FLAG tag, an SBP tag, and the like.
- HA hemagglutinin
- the tagged cell surface marker is modified to alter its activity, including but not limited to ATetherin-HA, ATetherin-FLAG, ATetherin-SBP and the like.
- the enrichment handle can bind affinity ligands ⁇ e.g., engineered to contain an HA tag, a FLAG tag, an SBP tag, and the like).
- the enrichment handle can be a heterologous cell surface receptor (a cell surface receptor not generally present in the cell type to be edited) or autologous cell surface antigen with an engineered epitope tag.
- the methods use an editing selection cassette, e.g., a GFP-to-BFP editing cassette.
- the disclosure also includes automated multi-module cell editing instruments with an enrichment module that performs enrichment methods including those described herein to increase the overall editing efficiency in a population of cells, e.g., mammalian cells.
- One exemplary automated multi-module cell editing instrument of the disclosure includes a housing configured to house all or some of the modules, a receptacle configured to receive cells, one or more receptacles configured to receive nucleic acids, an editing machinery introduction module configured to introduce the nucleic acids and/or proteins into the cells, a recovery module configured to allow the cells to recover after introduction of the editing machinery, an enrichment module for enrichment of cells that have received the editing nucleic acids and/or nuclease, an editing module configured to allow the introduced nucleic acids to edit nucleic acids in the cells, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a pre-programmed script.
- One exemplary automated multi-module cell editing instrument of the disclosure includes a housing configured to house all or some of the modules, a receptacle configured to receive cells and editing nucleic acids, an editing machinery introduction module configured to introduce the nucleic acids into the cells, a recovery module configured to allow the cells to recover after introduction of the editing machinery, an enrichment module for enrichment of cells that have received the editing nucleic acids and/or nuclease, an editing module configured to allow the introduced nucleic acids to edit nucleic acids in the cells, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a pre-programmed script.
- One exemplary automated multi-module cell editing instrument of the disclosure includes a housing configured to house some or all of the modules, a receptacle configured to receive cells, at least one receptacle configured to receive nucleic acids for editing, a growth module configured to grow the cells, an editing machinery introduction module comprising a flow-through electroporator to introduce editing nucleic acids into the cells, an enrichment module for enrichment of cells that have received the editing nucleic acids and/or nuclease, an editing module configured to allow the editing nucleic acids to edit nucleic acids in the cells, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a pre-programmed script.
- One exemplary automated multi-module cell editing instrument of the disclosure includes a housing configured to house some or all of the modules, a receptacle configured to receive cells and editing nucleic acids, a growth module configured to grow the cells, a filtration module configured to concentrate the cells and render the cells electrocompetent, an editing machinery introduction module comprising a flow-through electroporator to introduce editing nucleic acids into the cells, an enrichment module for enrichment of cells that have received the editing nucleic acids, an editing module configured to allow the cells to recover after electroporation and to allow the nucleic acids to edit the cells, and a processor configured to operate the automated multi-module cell editing instrument based on user input.
- the nucleic acids and/or cells are contained within a reagent cartridge, which is introduced into a receptacle of the instrument.
- a reagent cartridge for use with the present disclosure are described, e.g., in USPNs 10,376,889, 10,478,822, and 10,406,525, which are incorporated by reference herein for all purposes.
- the methods described herein enable the user to obtain a population of cells with a much higher proportion of cells with precise, intended edits and fewer unedited and/or imprecisely edited cells.
- the present methods can result in 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more intended edits within a cell population.
- the disclosure provides cell libraries created using the editing methods described herein in the disclosure.
- the disclosure provides cell libraries created using an automated editing system for nickase-directed genome editing, wherein the system comprises a housing, a receptacle configured to receive cells and one or more rationally designed nucleic acids comprising sequences to facilitate nickase-directed genome editing events in the cells, a transformation unit for introduction of the nucleic acid(s) into the cells, an editing unit for allowing the nickase-directed genome editing events to occur in the cells, an enrichment module, and a processor-based system configured to operate the instrument based on user input, where the nickase-directed genome editing events created by the automated system result in a cell library comprising individual cells with rationally designed edits.
- the disclosure provides cell libraries created using an automated editing system for nickase-directed genome editing, wherein the system comprises a housing, a cell receptacle configured to receive cells, a nucleic acid receptacle configured to receive one or more rationally designed nucleic acids comprising sequences to facilitate nickase-directed genome editing events in the cells, a transformation unit for introduction of the nucleic acid(s) into the cells, an editing unit for allowing the nickase-directed genome editing events to occur in the cells, and a processor based system configured to operate the instrument based on user input, where the nickase-directed genome editing events created by the automated system result in a cell library comprising individual cells with rationally designed edits.
- nucleic acid-guided nuclease techniques can be found in, e.g., Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, Appasani and Church (2016); and CRISPR: Methods and Protocols, Lindgren and Charpentier (2015); both of which are herein incorporated in their entirety by reference for all purposes.
- nucleic acid refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds.
- a nucleic acid includes a nucleotide sequence described as having a "percent complementarity” or “percent homology” to a specified second nucleotide sequence.
- a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence.
- the nucleotide sequence 3’-TCGA-5’ is 100% complementary to the nucleotide sequence 5’-AGCT-3’; and the nucleotide sequence 3’-TCGA-5’ is
- control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as a selected coding sequence is capable of being replicated, transcribed and — for some components — translated in an appropriate host cell.
- donor DNA or "donor nucleic acid” refers to nucleic acid that is designed to introduce a DNA sequence modification (insertion, deletion, substitution) into a locus (e.g., a target genomic DNA sequence or cellular target sequence) by homologous recombination using nucleic acid-guided nucleases.
- a locus e.g., a target genomic DNA sequence or cellular target sequence
- the donor DNA must have sufficient homology to the regions flanking the “cut site” or site to be edited in the genomic target sequence. The length of the homology arm(s) will depend on, e.g., the type and size of the modification being made.
- the donor DNA will have two regions of sequence homology (e.g., two homology arms) to the genomic target locus.
- an "insert" region or “DNA sequence modification” region the nucleic acid modification that one desires to be introduced into a genome target locus in a cell — will be located between two regions of homology.
- the DNA sequence modification may change one or more bases of the target genomic DNA sequence at one specific site or multiple specific sites.
- a change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the genomic target sequence.
- a deletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the genomic target sequence.
- guide nucleic acid or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.
- homologous region or “homology arm” refers to a region on the donor DNA with a certain degree of homology with the target genomic DNA sequence. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
- nickase refers to a nuclease that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence.
- operably linked refers to an arrangement of elements where the components so described are configured so as to perform their usual function.
- control sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence.
- the control sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence.
- intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
- such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation.
- the terms "protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.
- a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible.
- selectable marker refers to a gene introduced into a cell, which confers a trait suitable for artificial selection.
- General use selectable markers are well-known to those of ordinary skill in the art.
- selectable markers can use means that deplete a cell population to enrich for editing, and include ampicillin/carbenicillin, kanamycin, chloramphenicol, nourseothricin N-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418 or other selectable markers may be employed.
- selectable markers include physical markers that confer a phenotype that can be utilized for physical or computations cell enrichment, e.g., optical selectable markers such as fluorescent proteins (e.g., green fluorescent protein, blue fluorescent protein) and cell surface handles.
- the term “specifically binds” as used herein includes an interaction between two molecules, e.g., an engineered peptide antigen and a binding target, with a binding affinity represented by a dissociation constant of about 10 -7 M, about 10 -8 M, about 10- 9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M, about 10 -13 M, about 10 -14 M or about 10 -15 M.
- target genomic DNA sequence refers to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome or episome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system.
- the target sequence can be a genomic locus or extrachromosomal locus.
- variant may refer to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties.
- a typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
- a variant of a polypeptide may be a conservatively modified variant.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non- natural amino acid).
- a variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.
- a “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell.
- Vectors are typically composed of DNA, although RNA vectors are also available.
- Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.
- the term “editing vector” includes a coding sequence for a nuclease, a gRNA sequence to be transcribed, and a donor DNA sequence. In other embodiments, however, two vectors — an engine vector comprising the coding sequence for a nuclease, and an editing vector, comprising the gRNA sequence to be transcribed and the donor DNA sequence — may be used.
- FIGs. 1A-1C depict an automated multi-module instrument and components thereof with which to practice the recursive editing methods as taught herein.
- FIG. 2A depicts one embodiment of a rotating growth vial for use with the cell growth module described herein.
- FIG. 2B illustrates a perspective view of one embodiment of a rotating growth vial in a cell growth module.
- FIG. 2C depicts a cutaway view of the cell growth module from FIG.2B.
- FIG.2D illustrates the cell growth module of FIG. 2B coupled to LED, detector, and temperature regulating components.
- FIG. 3 A is a model of tangential flow filtration used in the TFF device presented herein.
- FIG. 3B depicts a top view of a lower member of one embodiment of an exemplary TFF device.
- FIG. 3C depicts a top view of upper and lower members and a membrane of an exemplary TFF device.
- FIG. 3 A is a model of tangential flow filtration used in the TFF device presented herein.
- FIG. 3B depicts a top view of a lower member of one embodiment of an exemplary TFF device.
- FIG. 3C depict
- FIG. 3D depicts a bottom view of upper and lower members and a membrane of an exemplary TFF device.
- FIGs. 3E - 3K depict various views of yet another embodiment of a TFF module having fluidically coupled reservoirs.
- FIG. 3L is an exemplary pneumatic architecture diagram for the TFF module described in relation to FIGs. 3E - 3K.
- FIGs. 4A shows a flow-through electroporation device exemplary (here, there are six such devices co-joined).
- FIG. 4B is a top view of one embodiment of an exemplary flow-through electroporation device.
- FIG. 4C depicts a top view of a cross section of the electroporation device of FIG. 4C.
- FIG. 4D is a side view cross section of a lower portion of the electroporation devices of FIGs. 4C and 4D.
- FIGs. 5A and 5B depict the structure and components of one embodiment of a reagent cartridge.
- FIG. 6 is a simplified block diagram of an embodiment of an exemplary automated multi-module cell processing instrument.
- FIG. 7 is a diagram showing a first set of exemplary workflows for carrying out editing and selection protocols of the disclosure.
- FIG. 8 is a diagram showing a second set of exemplary workflows for carrying out editing and selection protocols of the disclosure.
- FIG. 9 is a diagram showing a first set of exemplary workflows for carrying out CREATE Fusion Editing protocols of the disclosure.
- FIG. 10 is a diagram showing a second set of exemplary workflows for carrying out CREATE Fusion protocols of the disclosure.
- FIG. 11 is a diagram showing potential mechanism for editing using a fusion protein with reverse transcriptase activity over multiple cell cycles.
- FIG. 12 is a diagram illustrating exemplary elements in a plasmid structure used for the GFP expression assay.
- FIGs. 13A and 13B are plots showing the delivery of Nuclease-GFP expression cassettes as monitored by FACS.
- FIGs. 14A and 14B are plots showing GFP to BFP conversion for phenotypic assessment of NHEJ and HDR-mediated editing.
- FIG. 15 is a plot showing differential expression levels of a Thyl.2 reporter expressed from a GFP to BFP editing cassette.
- FIGs. 16A - 16E are a series of plots showing the effects of the enrichment
- FIG. 17 is a bar graph showing comparable enrichment of cell populations with higher editing rates (NHEJ and HDR) by either FACS or MACS.
- FIG. 18 is a bar graph showing ATetherin-HA Editing Cassette enriched editing demonstrated using FACS sorted cells.
- FIGs. 19A and 19B are a graph and table showing how MACS bead concentrations during enrichment affects the relative proportions of Thy 1.2 and
- FlGs. 20A and 20B are a graph and table showing how MACS bead concentrations during enrichment affect the relative proportions of ATetherin-HA enriched cells.
- FIG. 21 is a bar graph showing edit rates for cells enriched using various amounts of Thy 1.2-specific MACS beads.
- FIG.22 is a bar graph showing analysis post enrichment for cells expressing high levels of the ATetherin-HA reporter in HAP1.
- FIG. 23 is a bar graph showing enrichment of cells with higher knock-in editing rates at the DNMT3b gene using FACS enrichment techniques.
- FIG. 24 shows the designs of the CFE editing constructs CFE2.1 and CFE
- FIG. 25 shows the designs of various gRNAs that include the 13 bp TY-to- SH edit or a second region of 13 bp that is complementary to the nicked EGFP DNA sequence.
- FIG. 26 is a diagram showing the basic protocol for editing using the CREATE Fusion Editing cassettes of FIG. 25 in comparison to direct nuclease editing.
- FlGs. 27A-27D are graphs showing the editing of GFP-to-BFP HEK293T cells using various protocols.
- FIG. 28 is a diagram showing the basic protocol for CREATE Fusion Editing in conjunction with FACS selection.
- FIG. 29 is a graph showing the level of dsRed-Lo and dsRed-High cells resulting from editing with MAD7 nuclease editing versus CREATE Fusion Editing.
- FIG. 30 is a plot showing the differential expression levels of dsRed in the edited cell populations.
- FIG.31 is a bar graph showing dsRed editing for MAD7 or CREATE Fusion
- FIG. 32 is a diagram showing the basic protocol for CREATE Fusion Editing using a single gRNA.
- FlGs. 33A - 33C are bar graphs showing the editing efficiencies of using the CREATE Fusion constructs CFE2.1 and CFE2.2 with Lenti viral delivery.
- FIGs. 34A and 34B are bar graphs comparing the editing efficiencies of using the CREATE Fusion construct CFE2.2 versus MAD7 editing, both with lentiviral delivery.
- FIGs. 35A and 35B are figures showing exemplary strategies for using a CREATE fusion editing system with a tracking or recording technology.
- This disclosure is directed to methods and instruments for improving precise editing in a population of cells.
- Various cellular mechanisms may be used in the editing process, including non-homologous end joining (NHEJ) repair, base editing, microhomology-directed repair (MMEJ) and/or homology-directed repair (HDR).
- NHEJ non-homologous end joining
- MMEJ microhomology-directed repair
- HDR homology-directed repair
- the methods and instruments improve editing via homology- directed repair (HDR); accordingly, in specific aspects, the disclosure provides methods of improving HDR in mammalian cells.
- the disclosure provides methods of improving HDR in human cells.
- the disclosure provides methods of improving HDR in human pluripotent cells, e.g., induced pluripotent stem cells.
- the disclosure provides enrichment of co-introduced nucleic acids for the enrichment of cells that have received the editing components necessary for nucleic acid-directed editing, e.g., using specific selection of cells that have been transfected with a plasmid containing a nucleic acid encoding a donor nucleic acid and/or a guide nucleic acid, and optionally a nuclease.
- the disclosure is directed to automated methods of increasing editing efficiencies using co-introduction of nucleic acids encoding editing machinery and a cell surface selection handle.
- the co-introduction of nucleic acids occurs in a multi-module automated instrument, as described in more detail herein.
- the disclosure provides methods of improving homology- directed repair (HDR) using proteins that are a combination of an RNA-directed nuclease and an enzymatic activity from a different protein, e.g., replication inhibition, reverse transcriptase activity, transcription enhancement activity, and the like.
- these nuclease fusion proteins have a nickase function, and thus result in a nick on a single strand of the DNA to be edited instead of a double stranded break.
- the editing nuclease fusion proteins can be used with editing nucleic acids such as those found, e.g., in U.S. Pat No. 9,982,278 and related patents.
- nucleic acids encoding a gRNA comprising a region complementary to a target region of a nucleic acid in one or more cells covalently linked to an editing cassette comprising a region homologous to the target region in the one or more cells with a mutation of at least one nucleotide relative to the target region in the one or more cells.
- These nucleic acids can optionally include a protospacer and/or a barcode.
- the editing methods can involve one or more sets of these nucleic acids, and result in two or more nicks in the target region for the intended edit. Examples of such methods include, but are not limited to, those described in Liu et al (Nature, 2019 Dec; 576(7785): 149-157).
- CREATE Fusion Editing is a novel technique that uses a nuclease editing enzyme having nickase activity in conjunction with one or more nucleic acids to facilitate editing.
- CREATE Fusion Editing methods utilize an editing fusion protein (e.g., a protein having CRISPR targeting activity and reverse transcriptase activity) and a nucleic acid encoding one or more gRNAs comprising a region complementary to a target region of a nucleic acid.
- the one or more gRNAs are covalently linked to an editing cassette comprising a region homologous to the target region having a mutation of at least one nucleotide relative to the target region for the intended edit in the one or more cells.
- the nucleic acid may further comprise a protospacer adjacent motif (PAM) mutation and/or a barcode indicative of the intended mutation in the target region.
- PAM protospacer adjacent motif
- an edit in the nuclease binding seed region can be utilized to render a site nuclease resistant, preventing additional cutting using the nuclease (e.g., a nuclease fusion protein containing nicking activity)
- the CREATE Fusion methods can utilize a fusion protein having nickase activity and a single gRNA to achieve high efficiency editing, two-fold or more over the techniques taught in Liu et al, supra.
- a single nick in the target region the methods of the present disclosure were able to achieve editing efficiencies of over 20%, including precise editing rates of up to 45%, in mammalian cells without enrichment.
- the single nick system disclosed herein which was able to achieve the high levels of editing efficiency previously described only utilizing a dual nick system.
- the editing machinery can be allowed to persist for several cell divisions. As shown in FIG. 9, this editing cycle in the cell population allows a higher percentage of the cells to be edited using the introduced CREATE Fusion Editing machinery.
- compositions and methods described herein are employed to perform nuclease-directed genome editing to introduce desired edits to a population of mammalian cells.
- a single edit is introduced in a single round of editing.
- multiple edits are introduced in a single round of editing using simultaneous editing, e.g., the introduction of two or more edits on a single vector.
- recursive cell editing is performed where edits are introduced in successive rounds of editing.
- a nucleic acid-guided nuclease complexed with an appropriate synthetic guide nucleic acid in a cell can cut the genome of the cell at a desired location.
- the guide nucleic acid helps the nucleic acid-guided nuclease recognize and cut the DNA at a specific target sequence.
- the nucleic acid-guided nuclease may be programmed to target any DNA sequence for cleavage as long as an appropriate protospacer adjacent motif (PAM) is nearby.
- PAM protospacer adjacent motif
- the nucleic acid-guided nuclease editing system may use two separate guide nucleic acid molecules that combine to function as a guide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).
- a guide nucleic acid e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).
- the guide nucleic acid is a single guide nucleic acid construct that includes both 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid- guided nuclease.
- a guide nucleic acid e.g., gRNA
- a guide nucleic acid complexes with a compatible nucleic acid-guided nuclease and can then hybridize with a target sequence, thereby directing the nuclease to the target sequence.
- a guide nucleic acid can be DNA or RNA; alternatively, a guide nucleic acid may comprise both DNA and RNA.
- a guide nucleic acid may comprise modified or non-naturally occurring nucleotides.
- the gRNA may be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or the coding sequence may and preferably does reside within an editing cassette.
- editing cassettes see, e.g., USPNs 10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; and 10,435,715; and USSN 16/275,465, filed 14 February 2019, all of which are incorporated by reference herein for all purposes.
- a guide nucleic acid comprises a guide sequence, where the guide sequence is a polynucleotide sequence having sufficient complementarity (i.e homology) with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a complexed nucleic acid-guided nuclease to the target sequence.
- the degree of complementarity between a guide sequence and the corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
- Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences.
- a guide sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20 nucleotides in length.
- the gRN A/nuclease complex binds to a target sequence as determined by the guide RNA, and the nuclease recognizes a protospacer adjacent motif (PAM) sequence adjacent to the target sequence.
- the target sequence can be any polynucleotide endogenous or exogenous to the mammalian cell, or in vitro.
- the target sequence can be a polynucleotide residing in the nucleus of the mammalian cell.
- a target sequence can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, a control sequence, or “junk” DNA).
- a gene product e.g., a protein
- a non-coding sequence e.g., a regulatory polynucleotide, an intron, a PAM, a control sequence, or “junk” DNA.
- the guide nucleic acid may be and preferably is part of an editing cassette that encodes the donor nucleic acid that targets a cellular target sequence. Alternatively, the guide nucleic acid may not be part of the editing cassette and instead may be encoded on the editing vector backbone.
- a sequence coding for a guide nucleic acid can be assembled or inserted into a vector backbone first, followed by insertion of the donor nucleic acid in, e.g., an editing cassette.
- the donor nucleic acid in, e.g., an editing cassette can be inserted or assembled into a vector backbone first, followed by insertion of the sequence coding for the guide nucleic acid.
- the sequence encoding the guide nucleic acid and the donor nucleic acid are located together in a rationally-designed editing cassette and are simultaneously inserted or assembled via gap repair into a linear plasmid or vector backbone to create an editing vector.
- a PCR amplicon of the editing cassette can be used for editing.
- the target sequence is associated with a proto-spacer mutation (PAM), which is a short nucleotide sequence recognized by the gRN A/nuclease complex.
- PAM proto-spacer mutation
- the precise preferred PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5’ or 3’ to the target sequence.
- Engineering of the PAM-interacting domain of a nucleic acid-guided nuclease may allow for alteration of PAM specificity, improve target site recognition fidelity, decrease target site recognition fidelity, or increase the versatility of a nucleic acid-guided nuclease.
- the genome editing of a cellular target sequence both introduces a desired DNA change to a cellular target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a proto-spacer mutation (PAM) region in the cellular target sequence. Rendering the PAM at the cellular target sequence inactive precludes additional editing of the cell genome at that cellular target sequence, e.g., upon subsequent exposure to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in later rounds of editing.
- a desired DNA change e.g., the genomic DNA of a cell
- PAM proto-spacer mutation
- cells having the desired cellular target sequence edit and an altered PAM can be selected for by using a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid complementary to the cellular target sequence.
- Cells that did not undergo the first editing event will be cut rendering a double-stranded DNA break, and thus will not continue to be viable.
- the cells containing the desired cellular target sequence edit and PAM alteration will not be cut, as these edited cells no longer contain the necessary PAM site and will continue to grow and propagate.
- a polynucleotide sequence encoding the nucleic acid-guided nuclease can be codon optimized for expression in particular mammalian cell types, such as stem cells.
- the choice of nucleic acid-guided nuclease to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence.
- Nucleases of use in the methods described herein include but are not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.
- the nuclease is encoded by a DNA sequence on a vector and optionally is under the control of an inducible promoter.
- the promoter may be separate from but the same as the promoter controlling transcription of the guide nucleic acid; that is, a separate promoter drives the transcription of the nuclease and guide nucleic acid sequences but the two promoters may be the same type of promoter.
- the promoter controlling expression of the nuclease may be different from the promoter controlling transcription of the guide nucleic acid; that is, e.g., the nuclease may be under the control of, e.g., the pTEF promoter, and the guide nucleic acid may be under the control of the, e.g., pCYCl promoter.
- the donor nucleic acid comprising homology to the cellular target sequence.
- the donor nucleic acid is on the same vector and even in the same editing cassette as the guide nucleic acid and preferably is (but not necessarily is) under the control of the same promoter as the editing gRNA (that is, a single promoter driving the transcription of both the editing gRNA and the donor nucleic acid).
- the donor nucleic acid is designed to serve as a template for homologous recombination with a cellular target sequence nicked or cleaved by the nucleic acid-guided nuclease as a part of the gRN A/nuclease complex.
- a donor nucleic acid polynucleotide may be of any suitable length, such as about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides in length, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and up to 20 kb in length if combined with a dual gRNA architecture as described in USSN 62/869,240, filed 01 July 2019.
- the donor nucleic acid can be provided as an oligonucleotide of between 20-300 nucleotides, more preferably between 50-250 nucleotides.
- the donor nucleic acid comprises a region that is complementary to a portion of the cellular target sequence (e.g., a homology arm).
- the donor nucleic acid overlaps with (is complementary to) the cellular target sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides.
- the donor nucleic acid comprises two homology arms (regions complementary to the cellular target sequence) flanking the mutation or difference between the donor nucleic acid and the cellular target sequence.
- the donor nucleic acid comprises at least one mutation or alteration compared to the cellular target sequence, such as an insertion, deletion, modification, or any combination thereof compared to the cellular target sequence.
- the donor nucleic acid can be provided as part of a rationally-designed editing cassette, which is inserted into an editing plasmid backbone where the editing plasmid backbone may comprise a promoter to drive transcription of the editing gRNA and the donor DNA when the editing cassette is inserted into the editing plasmid backbone.
- a single rationally- designed editing cassette may comprise two to several editing gRNA/donor DNA pairs, where each editing gRNA is under the control of separate different promoters, separate like promoters, or where all gRNAs/donor nucleic acid pairs are under the control of a single promoter.
- the promoter driving transcription of the editing gRNA and the donor nucleic acid is optionally an inducible promoter.
- an editing cassette may comprise one or more primer sites.
- the primer sites can be used to amplify the editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the editing cassette.
- the editing cassette may comprise a barcode.
- a barcode is a unique DNA sequence that corresponds to the donor DNA sequence such that the barcode can identify the edit made to the corresponding cellular target sequence.
- the barcode typically comprises four or more nucleotides.
- the editing cassettes comprise a collection or library editing gRNAs and of donor nucleic acids representing, e.g., gene- wide or genome-wide libraries of editing gRNAs and donor nucleic acids.
- an editing vector or plasmid encoding components of the nucleic acid-guided nuclease system further encodes a nucleic acid- guided nuclease comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs, particularly as an element of the nuclease sequence.
- NLSs nuclear localization sequences
- the engineered nuclease comprises NLSs at or near the amino-terminus, NLSs at or near the carboxy-terminus, or a combination.
- Cells with a stably integrated genomic copy of the GFP gene can enable phenotypic detection of genomic edits of different classes (NHEJ, HDR, no edit) by flow cytometry, fluorescent cell imaging, or genotypic detection by sequencing of the genome-integrated GFP gene.
- Lack of editing, or perfect repair of cut events in the GFP gene result in cells that remain GFP-positive.
- Cut events that are repaired by the Non-Homologous End-Joining (NHEJ) pathway often result in nucleotide insertion or deletion events (Indel).
- NHEJ Non-Homologous End-Joining
- Indel nucleotide insertion or deletion events
- These Indel edits often result in frame-shift mutations in the coding sequence that cause loss of GFP gene expression and fluorescence.
- Cut events that are repaired by the Homology-Directed Repair (HDR) pathway using the GFP to BFP HDR donor as a repair template result in conversion of the cell fluorescence profile
- the editing cassette used was a plasmid that mediates expression of a gRNA that targets the nuclease to a specific DNA sequence.
- the editing cassette plasmid can also have a DNA sequence (e.g., HDR donor) to provide a template for targeted insertions, deletions, or nucleotide swaps proximal to the nuclease-targeted cut site.
- the editing cassette plasmid expresses a gRNA targeting a stably integrated genomic copy of the GFP gene and provides an HDR donor that mediates nucleotide swaps which convert the amino acid coding sequence of GFP to that of BFP.
- RNA-guided nuclease e.g., Cas9, Cpfl, MAD7
- a nuclease-encoding expression plasmid nuclease-encoding mRNA, recombinant nuclease protein, or by generation of a nuclease-expressing stable cell line.
- the MAD7 nuclease was delivered by means of a nucleaseencoding expression plasmid.
- Editing cassette plasmid and nuclease can be delivered to the target cell by traditional mammalian cell transfection techniques.
- FIG. 1A depicts an exemplary automated multi-module cell processing instrument 100 to, e.g., perform one of the exemplary workflows comprising a split protein reporter system as described herein.
- the instrument 100 may be and preferably is designed as a stand-alone desktop instrument for use within a laboratory environment.
- the instrument 100 may incorporate a mixture of reusable and disposable components for performing the various integrated processes in conducting automated genome cleavage and/or editing in cells without human intervention.
- a gantry 102 providing an automated mechanical motion system (actuator) (not shown) that supplies XYZ axis motion control to, e.g., an automated (i.e., robotic) liquid handling system 158 including, e.g., an air displacement pipettor 132 which allows for cell processing among multiple modules without human intervention.
- an automated (i.e., robotic) liquid handling system 158 including, e.g., an air displacement pipettor 132 which allows for cell processing among multiple modules without human intervention.
- the air displacement pipettor 132 is moved by gantry 102 and the various modules and reagent cartridges remain stationary; however, in other embodiments, the liquid handling system 158 may stay stationary while the various modules and reagent cartridges are moved.
- reagent cartridges 110 comprising reservoirs 112 and editing machinery introduction module 130 (e.g., a flow-through electroporation device as described in detail in relation to FlGs.4A - 4D), as well as wash reservoirs 106, cell input reservoir 151 and cell output reservoir 153.
- the wash reservoirs 106 may be configured to accommodate large tubes, for example, wash solutions, or solutions that are used often throughout an iterative process.
- two of the reagent cartridges 110 comprise a wash reservoir 106 in FIG. 1A, the wash reservoirs instead could be included in a wash cartridge where the reagent and wash cartridges are separate cartridges.
- the reagent cartridge 110 and wash cartridge 104 may be identical except for the consumables (reagents or other components contained within the various inserts) inserted therein. Note that an exemplary reagent cartridge is illustrated in FIGs. 5A and 5B.
- the reagent cartridges 110 are disposable kits comprising reagents and cells for use in the automated multi-module cell processing/editing instrument 100.
- a user may open and position each of the reagent cartridges 110 comprising various desired inserts and reagents within the chassis of the automated multi-module cell editing instrument 100 prior to activating cell processing.
- each of the reagent cartridges 110 may be inserted into receptacles in the chassis having different temperature zones appropriate for the reagents contained therein.
- the robotic liquid handling system 158 including the gantry 102 and air displacement pipettor 132.
- the robotic handling system 158 may include an automated liquid handling system such as those manufactured by Tecan Group Ltd. of Mannedorf, Switzerland, Hamilton Company of Reno, NV (see, e.g., WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, CO. (see, e.g., US20160018427A1).
- Pipette tips may be provided in a pipette transfer tip supply (not shown) for use with the air displacement pipettor 132.
- Inserts or components of the reagent cartridges 110 are marked with machine-readable indicia (not shown), such as bar codes, for recognition by the robotic handling system 158.
- the robotic liquid handling system 158 may scan one or more inserts within each of the reagent cartridges 110 to confirm contents.
- machine-readable indicia may be marked upon each reagent cartridge 110, and a processing system (not shown, but see element 137 of FIG. IB) of the automated multi-module cell editing instrument 100 may identify a stored materials map based upon the machine-readable indicia.
- a processing system not shown, but see element 137 of FIG. IB
- a cell growth module comprises two cell growth vials 118, 120 (described in greater detail below in relation to FIGs. 2A - 2D). Additionally seen is the TFF module 122 (described above in detail in relation to FIGs. 3A - 3L). Additionally seen is an enrichment module 140. Also note the placement of three heatsinks 155.
- FIG. IB is a simplified representation of the contents of the exemplary multimodule cell processing instrument 100 depicted in FIG. 1A.
- Cartridge-based source materials such as in reagent cartridges 110
- the deck of the multi-module cell processing instrument 100 may include a protection sink such that contaminants spilling, dripping, or overflowing from any of the modules of the instrument 100 are contained within a lip of the protection sink.
- reagent cartridges 110 which are shown disposed with thermal assemblies 111 which can create temperature zones appropriate for different regions.
- one of the reagent cartridges also comprises a flow-through electroporation device 130 (FTEP), served by FTEP interface (e.g., manifold arm) and actuator 131.
- FTEP flow-through electroporation device 130
- TFF module 122 with adjacent thermal assembly 125, where the TFF module is served by TFF interface (e.g., manifold arm) and actuator 133.
- Thermal assemblies 125, 135, and 145 encompass thermal electric devices such as Peltier devices, as well as heatsinks, fans and coolers.
- the rotating growth vials 118, 120 are within a growth module 134, where the growth module is served by two thermal assemblies 135.
- An enrichment module is seen at 140, where the enrichment module is served by selection interface (e.g., manifold arm) and actuator 147.
- element 137 comprises electronics, such as circuit control boards, high-voltage amplifiers, power supplies, and power entry; as well as pneumatics, such as pumps, valves and sensors.
- FIG. 1C illustrates a front perspective (door open) view of multi-module cell processing instrument 100 for use in as a desktop version of the automated multimodule cell editing instrument 100.
- a chassis 190 may have a width of about 24—48 inches, a height of about 24-48 inches and a depth of about 24-48 inches.
- Chassis 190 may be and preferably is designed to hold all modules and disposable supplies used in automated cell processing and to perform all processes required without human intervention; that is, chassis 190 is configured to provide an integrated, stand-alone automated multi-module cell processing instrument.
- chassis 190 includes touch screen display 101, cooling grate 164, which allows for air flow via an internal fan (not shown).
- the touch screen display provides information to a user regarding the processing status of the automated multi-module cell editing instrument 100 and accepts inputs from the user for conducting the cell processing.
- the chassis 190 is lifted by adjustable feet 170a, 170b, 170c and 170d (feet 170a- 170c are shown in this FIG. 1C). Adjustable feet 170a - 170d, for example, allow for additional air flow beneath the chassis 290.
- chassis 190 Inside the chassis 190, in some implementations, will be most or all of the components described in relation to FIGs. 1A and IB, including the robotic liquid handling system disposed along a gantry, reagent cartridges 110 including a flowthrough electroporation device, rotating growth vials 118, 120 in a cell growth module 134, a tangential flow filtration module 122, an enrichment module 140 as well as interfaces and actuators for the various modules.
- chassis 190 houses control circuitry, liquid handling tubes, air pump controls, valves, sensors, thermal assemblies (e.g., heating and cooling units) and other control mechanisms.
- FIG. 2A shows one embodiment of a rotating growth vial 200 for use with the cell growth device described herein configured to grow various cell types including microbial and mammalian cells lines and primary or generated stem cells (e.g., induced pluripotent stem cells, hematopoietic stem cells, embryonic stem cells and the like).
- the rotating growth vial is an optically-transparent container having an open end 204 for receiving liquid media and cells, a central vial region 206 that defines the primary container for growing cells, a tapered-to-constricted region 218 defining at least one light path 210, a closed end 216, and a drive engagement mechanism 212.
- the rotating growth vial has a central longitudinal axis 220 around which the vial rotates, and the light path 210 is generally perpendicular to the longitudinal axis of the vial.
- the first light path 210 is positioned in the lower constricted portion of the tapered-to-constricted region 218.
- some embodiments of the rotating growth vial 200 have a second light path 208 in the tapered region of the tapered-to-constricted region 218. Both light paths in this embodiment are positioned in a region of the rotating growth vial that is constantly filled with the cell culture (cells + growth media) and is not affected by the rotational speed of the growth vial.
- the first light path 210 is shorter than the second light path 208 allowing for sensitive measurement of OD values when the OD values of the cell culture in the vial are at a high level (e.g., later in the cell growth process), whereas the second light path 208 allows for sensitive measurement of OD values when the OD values of the cell culture in the vial are at a lower level (e.g., earlier in the cell growth process).
- lip 202 which allows the rotating growth vial to be seated in a growth module (not shown) and further allows for easy handling for the user.
- the rotating growth vial has two or more “paddles” or interior features disposed within the rotating growth vial, extending from the inner wall of the rotating growth vial toward the center of the central vial region.
- the width of the paddles or features varies with the size or volume of the rotating growth vial, and may range from 1/20 to just over 1/3 the diameter of the rotating growth vial, or from 1/15 to 1/4 the diameter of the rotating growth vial, or from 1/10 to 1/5 the diameter of the rotating growth vial.
- the length of the paddles varies with the size or volume of the rotating growth vial, and may range from 4/5 to 1/4 the length of the main body of the rotating growth vial, or from 3/4 to 1/3 the length of the main body of the rotating growth vial, or from 1/2 to 1/3 the length of the main body of the rotating growth vial.
- the concentric rows of raised features or spiral configuration may be disposed upon a post or center structure of the rotating growth vial.
- the rotating growth vial may comprise 3, 4, 5, 6 or more paddles, and up to 20 paddles.
- the number of paddles will depend upon, e.g., the size or volume of the rotating growth vial.
- the paddles may be arranged symmetrically as single paddles extending from the inner wall of the vial into the interior of the vial, or the paddles may be symmetrically arranged in groups of 2, 3, 4 or more paddles in a group (for example, a pair of paddles opposite another pair of paddles) extending from the inner wall of the vial into the interior of the vial.
- the paddles may extend from the middle of the rotating growth vial out toward the wall of the rotating growth vial, from, e.g., a post or other support structure in the interior of the rotating growth vial.
- the drive engagement mechanism 212 engages with a motor (not shown) to rotate the vial.
- the motor drives the drive engagement mechanism 212 such that the rotating growth vial is rotated in one direction only, and in other embodiments, the rotating growth vial is rotated in a first direction for a first amount of time or periodicity, rotated in a second direction (i.e., the opposite direction) for a second amount of time or periodicity, and this process may be repeated so that the rotating growth vial (and the cell culture contents) are subjected to an oscillating motion.
- the first amount of time and the second amount of time may be the same or may be different.
- the amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or more minutes.
- the rotating growth vial in an early stage of cell growth may be oscillated at a first periodicity (e.g., every 60 seconds), and then a later stage of cell growth the rotating growth vial may be oscillated at a second periodicity (e.g., every one second) different from the first periodicity.
- a first periodicity e.g., every 60 seconds
- a second periodicity e.g., every one second
- the rotating growth vial 200 may be specifically tailored for the growth of particular cell types. For example, O 2 and/or CO 2 can be specifically monitored or controlled, and the rotating growth vial may be designed and OD measurement modified to be compatible with use of specific carrier substrates for growth of adherent cells.
- the rotating growth vial 200 may be reusable or, preferably, the rotating growth vial is consumable. In some embodiments, the rotating growth vial is consumable and is presented to the user pre-filled with growth medium, where the vial is hermetically sealed at the open end 204 with a foil seal.
- a medium-filled rotating growth vial packaged in such a manner may be part of a kit for use with a stand-alone cell growth device or with a cell growth module that is part of an automated multimodule cell processing instrument. To introduce cells into the vial, a user need only pipette up a desired volume of cells and use the pipette tip to punch through the foil seal of the vial.
- Open end 204 may optionally include an extended lip 202 to overlap and engage with the cell growth device (not shown).
- the rotating growth vial 200 may be tagged with a barcode or other identifying means that can be read by a scanner or camera that is part of the automated system (not shown).
- the volume of the rotating growth vial 200 and the volume of the cell culture (including growth medium) may vary greatly, but the volume of the rotating growth vial 200 must be large enough for the cell culture in the growth vial to get proper aeration while the vial is rotating.
- the volume of the rotating growth vial 200 may range from 1-250 ml, 2-100 ml, from 5-80 ml, 10-50 ml, or from 12-35 ml.
- the volume of the cell culture (cells + growth media) should be appropriate to allow proper aeration in the rotating growth vial.
- the volume of the cell culture should be approximately 10-85% of the volume of the growth vial or from 20-60% of the volume of the growth vial.
- the rotating growth vial 200 preferably is fabricated from a bio-compatible optically transparent material — or at least the portion of the vial comprising the light path(s) is transparent. Additionally, material from which the rotating growth vial is fabricated should be able to be cooled to about 4°C or lower and heated to about 55 °C or higher to accommodate both temperature-based cell assays and long-term storage at low temperatures. Further, the material that is used to fabricate the vial must be able to withstand temperatures up to 55 °C without deformation while spinning.
- FIGs. 2B-2D show an embodiment of a cell growth module 250 comprising a rotating growth vial 200.
- FIG. 2B is a perspective view of one embodiment of a cell growth device 250.
- FIG. 2C depicts a cut-away view of the cell growth device 250 from FIG. 2B.
- FIG. 2C depicts additional detail.
- upper bearing 242 and lower bearing 230 are shown positioned in main housing 226.
- Upper bearing 242 and lower bearing 230 support the vertical load of rotating growth vial 200.
- Lower housing 232 contains the drive motor 236.
- the cell growth device of FIG. 2C comprises two light paths: a primary light path 234, and a secondary light path 230.
- Light path 234 corresponds to light path 210 positioned in the constricted portion of the tapered-to- constricted portion of the rotating growth vial
- light path 230 corresponds to light path 208 in the tapered portion of the tapered-to-constricted portion of the rotating growth vial.
- Light paths 210 and 208 are not shown in FIG. 2C but may be seen in, e.g., FIG. 2A.
- the motor 236 used to rotate the rotating growth vial 200 in some embodiments is a brushless DC type drive motor with built-in drive controls that can be set to hold a constant revolution per minute (RPM) between 0 and about 3000 RPM.
- RPM revolution per minute
- other motor types such as a stepper, servo, brushed DC, and the like can be used.
- the motor 206 may also have direction control to allow reversing of the rotational direction, and a tachometer to sense and report actual RPM.
- Main housing 226, end housings 222 and lower housing 232 of the cell growth device 250 may be fabricated from any suitable, robust material including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures or portions thereof can be created through various techniques, e.g., metal fabrication, injection molding, creation of structural layers that are fused, etc.
- the rotating growth vial is envisioned in some embodiments to be reusable but preferably is consumable
- the other components of the cell growth device 250 are preferably reusable and can function as a stand-alone benchtop device or, as here, as a module in a multi-module cell processing system.
- the processor (not shown) of the cell growth system may be programmed with information to be used as a “blank” or control for the growing cell culture.
- a “blank” or control is a vessel containing cell growth medium only, which yields 100% transmittance and 0 OD, while the cell sample will deflect light rays and will have a lower percent transmittance and higher OD. As the cells grow in the media and become denser, transmittance will decrease and OD will increase.
- the processor of the cell growth system may be programmed to use wavelength values for blanks commensurate with the growth media typically used in mammalian cell culture.
- FIG. 2D illustrates a cell growth device as part of an assembly comprising the cell growth device of FIG.2B coupled to light source 290, detector 292, and thermal components 294.
- the rotating growth vial 200 is inserted into the cell growth device.
- Components of the light source 290 and detector 292 e.g., such as a photodiode with gain control to cover 5-log are coupled to the main housing of the cell growth device.
- the lower housing 232 that houses the motor that rotates the rotating growth vial is illustrated, as is one of the flanges 224 that secures the cell growth device to the assembly. Also illustrated is a Peltier device or thermoelectric cooler 294.
- thermal control is accomplished by attachment and electrical integration of the cell growth device 200 to the thermal device 294 via the flange 204 on the base of the lower housing 232.
- Thermoelectric coolers are capable of “pumping” heat to either side of a junction, either cooling a surface or heating a surface depending on the direction of current flow.
- a thermistor is used to measure the temperature of the main housing and then, through a standard electronic proportional- integral-derivative (PID) controller loop, the rotating growth vial 200 is controlled to approximately +/- 0.5 °C.
- PID proportional- integral-derivative
- a rear-mounted power entry module contains the safety fuses and the on-off switch, which when switched on powers the internal AC and DC power supplies (not shown) activating the processor.
- Measurements of optical densities (OD) at programmed time intervals are accomplished using a 600 nm Light Emitting Diode (LED) (not shown) that has been columnated through an optic into the lower constricted portion of the rotating growth vial which contains the cells of interest. The light continues through a collection optic to the detection system which consists of a (digital) gain-controlled silicone photodiode.
- LED Light Emitting Diode
- cells are inoculated (cells can be pipetted, e.g., from an automated liquid handling system or by a user) into pre-filled growth media of a rotating growth vial by piercing though the foil seal.
- the programmed software of the cell growth device sets the control temperature for growth, typically 30 °C, then slowly starts the rotation of the rotating growth vial.
- the cell/growth media mixture slowly moves vertically up the wall due to centrifugal force allowing the rotating growth vial to expose a large surface area of the mixture to a normal oxygen environment.
- the growth monitoring system takes either continuous readings of the OD or OD measurements at pre-set or pre-programmed time intervals.
- the software plots the measurements versus time to display a growth curve. If enhanced mixing is required, e.g., to optimize growth conditions, the speed of the vial rotation can be varied to cause an axial precession of the liquid, and/or a complete directional change can be performed at programmed intervals.
- the growth monitoring can be programmed to automatically terminate the growth stage at a predetermined OD, and then quickly cool the mixture to a lower temperature to inhibit further growth.
- One application for the cell growth device 250 is to constantly measure the optical density of a growing cell culture.
- One advantage of the described cell growth device is that optical density can be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5, 10, 15, 20, 3045, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 on minutes.
- OD optical density
- the cell growth device has been described in the context of measuring the optical density (OD) of a growing cell culture, it should, however, be understood by a skilled artisan given the teachings of the present specification that other cell growth parameters can be measured in addition to or instead of cell culture OD. For example, spectroscopy using visible, UV, or near infrared (NIR) light allows monitoring the concentration of nutrients and/or wastes in the cell culture.
- NIR near infrared
- spectroscopic measurements may be used to quantify multiple chemical species simultaneously.
- Nonsymmetric chemical species may be quantified by identification of characteristic absorbance features in the NIR.
- symmetric chemical species can be readily quantified using Raman spectroscopy.
- Many critical metabolites, such as glucose, glutamine, ammonia, and lactate have distinct spectral features in the IR, such that they may be easily quantified.
- the amount and frequencies of light absorbed by the sample can be correlated to the type and concentration of chemical species present in the sample.
- FT-NIR provides the greatest light penetration depth and can be used for thicker sample.
- FT-mid-IR provides information that is more easily discernible as being specific for certain analytes as these wavelengths are closer to the fundamental IR absorptions.
- FT-Raman is advantageous when interference due to water is to be minimized.
- Other spectral properties can be measured via, e.g., dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence.
- the cell growth device may include additional sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.
- FIGs. 3A - 3K depict variations on one embodiment of a cell concentration/buffer exchange cassette and module that utilizes tangential flow filtration and is configured for use with all cell types, including immortalized cell lines, primary cells and/or stem cells.
- a cell concentration device described herein operates using tangential flow filtration (TFF), also known as crossflow filtration, in which the majority of the feed flows tangentially over the surface of the filter thereby reducing cake (retentate) formation as compared to dead-end filtration, in which the feed flows into the filter.
- TMF tangential flow filtration
- Secondary flows relative to the main feed are also exploited to generate shear forces that prevent filter cake formation and membrane fouling thus maximizing particle recovery, as described below.
- FIG. 3A is a general model of tangential flow filtration.
- the TFF device operates using tangential flow filtration, also known as cross-flow filtration.
- FIG. 3A shows a system 390 with cells flowing over a membrane 394, where the feed flow of the cells 392 in medium or buffer is parallel to the membrane 394.
- TFF is different from dead-end filtration where both the feed flow and the pressure drop are perpendicular to a membrane or filter.
- TFF device 300 comprises a channel structure 316 comprising a flow channel 302b through which a cell culture is flowed.
- the channel structure 316 comprises a single flow channel 302b that is horizontally bifurcated by a membrane (not shown) through which buffer or medium may flow, but cells cannot.
- This particular embodiment comprises an undulating serpentine geometry 314 (i.e., the small “wiggles” in the flow channel 302) and a serpentine “zig-zag” pattern where the flow channel 302 crisscrosses the device from one end at the left of the device to the other end at the right of the device.
- the serpentine pattern allows for filtration over a high surface area relative to the device size and total channel volume, while the undulating contribution creates a secondary inertial flow to enable effective membrane regeneration preventing membrane fouling.
- an undulating geometry and serpentine pattern are exemplified here, other channel configurations may be used as long as the channel can be bifurcated by a membrane, and as long as the channel configuration provides for flow through the TFF module in alternating directions.
- portals 304 and 306 as part of the channel structure 316 can be seen, as well as recesses 308.
- Portals 304 collect cells passing through the channel on one side of a membrane (not shown) (the “retentate”), and portals 306 collect the medium (“filtrate” or “permeate”) passing through the channel on the opposite side of the membrane (not shown).
- recesses 308 accommodate screws or other fasteners (not shown) that allow the components of the TFF device to be secured to one another.
- the length 310 and width 312 of the channel structure 316 may vary depending on the volume of the cell culture to be grown and the optical density of the cell culture to be concentrated.
- the length 310 of the channel structure 316 typically is from 1 mm to 300 mm, or from 50 mm to 250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mm to 100 mm.
- the width of the channel structure 316 typically is from 1 mm to 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from 40 mm to 70 mm, or from 50 mm to 60 mm.
- the cross-section configuration of the flow channel 102 may be round, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape with generally straight sides, the cross section may be from about 10 ⁇ m to 1000 ⁇ m wide, or from 200 ⁇ m to 800 ⁇ m wide, or from 300 ⁇ m to 700 ⁇ m wide, or from 400 ⁇ m to 600 ⁇ m wide; and from about 10 ⁇ m to 1000 ⁇ m high, or from 200 ⁇ m to 800 ⁇ m high, or from 300 ⁇ m to 700 ⁇ m high, or from 400 ⁇ m to 600 ⁇ m high.
- the radius of the channel may be from about 50 ⁇ m to 1000 ⁇ m in hydraulic radius, or from 5 ⁇ m to 800 ⁇ m in hydraulic radius, or from 200 ⁇ m to 700 ⁇ m in hydraulic radius, or from 300 ⁇ m to 600 ⁇ m wide in hydraulic radius, or from about 200 to 500 ⁇ m in hydraulic radius.
- retentate and filtrate portals can on the same surface of the same member (e.g., upper or lower member), or they can be arranged on the side surfaces of the assembly.
- the TFF device/module described herein uses an alterating method for concentrating cells.
- the overall workflow for cell concentration using the TFF device/module involves flowing a cell culture or cell sample tangentially through the channel structure.
- the membrane bifurcating the flow channels retains the cells on one side of the membrane and allows unwanted medium or buffer to flow across the membrane into a filtrate side (e.g., lower member 320) of the device.
- a fixed volume of cells in medium or buffer is driven through the device until the cell sample is collected into one of the retentate portals 304, and the medium/buffer that has passed through the membrane is collected through one or both of the filtrate portals 306.
- All types of prokaryotic and eukaryotic cells can be grown in the TFF device.
- Adherent cells may be grown on beads or other cell scaffolds suspended in medium that flow through the TFF device.
- the retentate and filtrate portals collecting the cells and medium, respectively, for a given pass reside on the same end of TFF device/module 300 with fluidic connections arranged so that there are two distinct flow layers for the retentate and filtrate sides, but if the retentate portal 304 resides on the upper member of device/module 300 (that is, the cells are driven through the channel above the membrane and the filtrate (medium) passes to the portion of the channel below the membrane), the filtrate portal 306 will reside on the lower member of device/module 100 and vice versa (that is, if the cell sample is driven through the channel below the membrane, the filtrate (medium) passes to the portion of the channel above the membrane).
- This configuration can be seen more clearly in FTGs. 3C - 3D, where the retentate flows 360 from the retentate portals 304 and the filtrate flows 370 from the filtrate portals 306.
- the cell sample is collected by passing through the retentate portal 304 and into the retentate reservoir (not shown).
- the cell sample is passed again through the TFF device, this time in a flow direction that is reversed from the first pass.
- the cell sample is collected by passing through the retentate portal 304 and into retentate reservoir (not shown) on the opposite end of the device/module from the retentate portal 304 that was used to collect cells during the first pass.
- the medium/buffer that passes through the membrane on the second pass is collected through the filtrate portal 306 on the opposite end of the device/module from the filtrate portal 306 that was used to collect the filtrate during the first pass, or through both portals.
- This alternating process of passing the retentate (the concentrated cell sample) through the device/module is repeated until the cells have been concentrated to a desired volume, and both filtrate portals can be open during the passes to reduce operating time.
- buffer exchange may be effected by adding a desired buffer (or fresh medium) to the cell sample in the retentate reservoir, before initiating another “pass”, and repeating this process until the old medium or buffer is diluted and filtered out and the cells reside in fresh medium or buffer. Note that buffer exchange and cell concentration may (and typically do) take place simultaneously.
- FIG. 3C depicts a top view of upper (322) and lower (320) members of an exemplary TFF module. Again, portals 304 and 306 are seen.
- recesses such as the recesses 308 seen in FIG. 3B — provide a means to secure the components (upper member 322, lower member 320, and membrane 324) of the TFF device/membrane to one another during operation via, e.g., screws or other like fasteners.
- an adhesive such as a pressure sensitive adhesive, or ultrasonic welding, or solvent bonding, may be used to couple the upper member 322, lower member 320, and membrane 324 together.
- the retentate and filtrate portals on the right side of the device/module will collect cells (flow path at 360) and medium (flow path at 370), respectively, for the same pass.
- the retentate is collected from portals 304 on the top surface of the TFF device, and filtrate is collected from portals 306 on the bottom surface of the device.
- the cells are maintained in the TFF flow channel above the membrane 324, while the filtrate (medium) flows through membrane 324 and then through portals 306; thus, the top/retentate portals and bottom/filtrate portals configuration is practical.
- retentate and filtrate portals may be implemented such as positioning both the retentate and filtrate portals on the side (as opposed to the top and bottom surfaces) of the TFF device.
- the channel structure 302b can be seen on the bottom member 320 of the TFF device 300.
- retentate and filtrate portals can reside on the same of the TFF device.
- membrane or filter 324 Also seen in FIG. 3C is membrane or filter 324.
- Filters or membranes appropriate for use in the TFF device/module are those that are solvent resistant, are contamination free during filtration, and are able to retain the types and sizes of cells of interest.
- pore sizes can be as low as 0.2 ⁇ m, however for other cell types, the pore sizes can be as high as 5 ⁇ m.
- the pore sizes useful in the TFF device/module include filters with sizes from 0.20 ⁇ m, 0.21 ⁇ m, 0.22 ⁇ m, 0.23 ⁇ m, 0.24 ⁇ m, 0.25 ⁇ m, 0.26 ⁇ m, 0.27 ⁇ m, 0.28 ⁇ m, 0.29 ⁇ m, 0.30 ⁇ m, 0.31 ⁇ m, 0.32 ⁇ m, 0.33 ⁇ m, 0.34 ⁇ m, 0.35 ⁇ m, 0.36 ⁇ m, 0.37 ⁇ m, 0.38 ⁇ m, 0.39 ⁇ m, 0.40 ⁇ m, 0.41 ⁇ m, 0.42 ⁇ m, 0.43 ⁇ m, 0.44 ⁇ m, 0.45 ⁇ m, 0.46 ⁇ m, 0.47 ⁇ m, 0.48 ⁇ m, 0.49 ⁇ m, 0.50 ⁇ m and larger.
- the filters may be fabricated from any suitable non-reactive material including cellulose mixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glass fiber, or metal substrates as in the case of laser or electrochemical etching.
- CME cellulose mixed ester
- PC polycarbonate
- PVDF polyvinylidene fluoride
- PES polyethersulfone
- PTFE polytetrafluoroethylene
- nylon glass fiber
- glass fiber or metal substrates as in the case of laser or electrochemical etching.
- the TFF device shown in FIGs 3C and 3D do not show a seat in the upper 312 and lower 320 members where the filter 324 can be seated or secured (for example, a seat half the thickness of the filter in each of upper 312 and lower 320 members); however, such a seat is contemplated in
- FIG. 3D depicts a bottom view of upper and lower components of the exemplary TFF module shown in FIG. 3C.
- FIG. 3D depicts a bottom view of upper (322) and lower (320) components of an exemplary TFF module. Again portals 304 and 306 are seen. Note again that there is one retentate portal and one filtrate portal on each end of the device/module. The retentate and filtrate portals on the left side of the device/module will collect cells (flow path at 360) and medium (flow path at 370), respectively, for the same pass. Likewise, the retentate and filtrate portals on the right side of the device/module will collect cells (flow path at 360) and medium (flow path at 370), respectively, for the same pass. In FIG.
- the channel structure 302a can be seen on the upper member 322 of the TFF device 300.
- the channel structure 302 of the upper 322 and lower 320 members (302a and 302b, respectively) mate to create the flow channel with the membrane 324 positioned horizontally between the upper and lower members of the flow channel thereby bifurcating the flow channel.
- Medium exchange (during cell growth) or buffer exchange (during cell concentration or rendering the cells competent) is performed on the TFF device/module by adding fresh medium to growing cells or a desired buffer to the cells concentrated to a desired volume; for example, after the cells have been concentrated at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200- fold or more.
- a desired exchange medium or exchange buffer is added to the cells either by addition to the retentate reservoir or thorough the membrane from the filtrate side and the process of passing the cells through the TFF device 300 is repeated until the cells have been grown to a desired optical density or concentrated to a desired volume in the exchange medium or buffer.
- the exchange buffer may comprise, e.g., glycerol or sorbitol thereby rendering the cells competent for transformation in addition to decreasing the overall volume of the cell sample.
- the TFF device 300 may be fabricated from any robust material in which channels (and channel branches) may be milled including stainless steel, silicon, glass, aluminum, or plastics including cyclic-olefin copolymer (COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, and copolymers of these and other polymers. If the TFF device/module is disposable, preferably it is made of plastic.
- the material used to fabricate the TFF device/module is thermally-conductive so that the cell culture may be heated or cooled to a desired temperature.
- the TFF device is formed by precision mechanical machining, laser machining, electro discharge machining (for metal devices); wet or dry etching (for silicon devices); dry or wet etching, powder or sandblasting, photostructuring (for glass devices); or thermoforming, injection molding, hot embossing, or laser machining (for plastic devices) using the materials mentioned above that are amenable to this mass production techniques.
- FIG. 3E depicts a configuration of an upper (retentate) member 3022 (on left), a membrane or filter 3024 (middle), and a lower (permeate/filtrate) member 3020 (on the right).
- the retentate member 3022 is no longer “upper” and the permeate/filtrate member 3020 is no longer “lower”, as the retentate member 3022 and permeate/filtrate member 3020 are coupled side-to-side as seen in FIGs. 3J and 3K.
- FIG. 3E depicts a configuration of an upper (retentate) member 3022 (on left), a membrane or filter 3024 (middle), and a lower (permeate/filtrate) member 3020 (on the right).
- the retentate member 3022 is no longer “upper” and the permeate/filtrate member 3020 is no longer “lower”, as the retentate member 3022 and permeate/filtrate member 3020 are coupled side-to-side as seen
- retentate member 3022 comprises a tangential flow channel 3002, which has a serpentine configuration that initiates at one lower comer of retentate member 3022 — specifically at retentate port 3028 — traverses across and up then down and across retentate member 3022, ending in the other lower comer of retentate member 3022 at a second retentate port 3028.
- energy director 3091 also seen on retentate member 3022 is energy director 3091, which circumscribes the region where membrane or filter 3024 is seated. Energy director 3091 in this embodiment mates with and serves to facilitate ultrasonic wending or bonding of retentate member 3022 with permeate/filtrate member 3020 via the energy director component on permeate/filtrate member 3020.
- membrane or filter 3024 has through- holes for retentate ports 3028, which is configured to seat within the circumference of energy directors 3091 between the retentate member 3022 and the permeate/filtrate member 3020.
- Permeate/ filtrate member 3020 comprises, in addition to energy director 3091, through-holes for retentate port 3028 at each bottom comer (which mate with the through-holes for retentate ports 3028 at the bottom comers of membrane 3024 and retentate ports 3028 in retentate member 3022), as well as a tangential flow channel 3002 and a single permeate/filtrate port 3026 positioned at the top and center of permeate/filtrate member 3020.
- the tangential flow channel 3002 structure in this embodiment has a serpentine configuration and an undulating geometry, although other geometries may be used.
- the length of the tangential flow channel is from 10 mm to 1000 mm, from 60 mm to 200 mm, or from 80 mm to 100 mm.
- the width of the channel structure is from 10 mm to 120 mm, from 40 mm to 70 mm, or from 50 mm to 60 mm.
- the cross section of the tangential flow channel 1202 is rectangular.
- the cross section of the tangential flow channel 1202 is 5 ⁇ m to 1000 ⁇ m wide and 5 ⁇ m to 1000 ⁇ m high, 300 ⁇ m to 700 ⁇ wide and 300 ⁇ to 700 ⁇ high, or 400 ⁇ to 600 ⁇ wide and 400 ⁇ m to 600 ⁇ high.
- the cross section of the tangential flow channel 1202 is circular, elliptical, trapezoidal, or oblong, and is 100 ⁇ m to 1000 ⁇ m in hydraulic radius, 300 ⁇ m to 700 ⁇ m in hydraulic radius, or 400 ⁇ m to 600 ⁇ m in hydraulic radius.
- FIG. 3F is a side perspective view of a reservoir assembly 3050. The embodiment of FIG.
- Reservoir assembly 3050 comprises retentate reservoirs 3052 on either side of a single permeate reservoir 3054.
- Retentate reservoirs 3052 are used to contain the cells and medium as the cells are transferred through the cell concentration/growth device or module and into the retentate reservoirs during cell concentration and/or growth.
- Permeate/filtrate reservoir 3054 is used to collect the filtrate fluids removed from the cell culture during cell concentration, or old buffer or medium during cell growth.
- buffer or medium is supplied to the permeate/filtrate member from a reagent reservoir separate from the device module. Additionally seen in FIG.
- 3F are grooves 3032 to accommodate pneumatic ports (not seen), permeate/filtrate port 3026, and retentate port through-holes 3028.
- the retentate reservoirs are fluidically coupled to the retentate ports 3028, which in turn are fluidically coupled to the portion of the tangential flow channel disposed in the retentate member (not shown).
- the permeate/filtrate reservoir is fluidically coupled to the permeate/filtrate port 3026 which in turn are fluidically coupled to the portion of the tangential flow channel disposed in permeate/filtrate member (not shown), where the portions of the tangential flow channels are bifurcated by membrane (not shown).
- up to 120 mL of cell culture can be grown and/or filtered, or up to 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL of cell culture can be grown and/or concentrated.
- FIG. 3G depicts a top-down view of the reservoir assembly 3050 shown in FIG. 3F
- FIG. 3H depicts a cover 3044 for reservoir assembly 3050 shown in FIG. 3F
- 31 depicts a gasket 3045 that in operation is disposed on cover 3044 of reservoir assembly 3050 shown in FIG. 3F
- FIG. 3G is a top-down view of reservoir assembly 3050, showing two retentate reservoirs 3052, one on either side of permeate reservoir 3054.
- FIG. 3H depicts a cover 3044 that is configured to be disposed upon the top of reservoir assembly 3050. Cover 3044 has round cut-outs at the top of retentate reservoirs 3052 and permeate/filtrate reservoir 3054.
- FIG. 31 depicts a gasket 3045 that is configured to be disposed upon the cover 3044 of reservoir assembly 3050. Seen are three fluid transfer ports 3042 for each retentate reservoir 3052 and for permeate/filtrate reservoir 3054. Again, three pneumatic ports 3030, for each retentate reservoir 3052 and for permeate/filtrate reservoir 3054, are shown.
- FIGs 3J depicts an embodiment of assembled TFF module 3000. Note that in this embodiment of a TFF module the retentate member 3022 is no longer “upper”, and the permeate/filtrate member 3020 is no longer “lower”, as the retentate member 3022 and permeate/filtrate member 3020 are coupled side-to-side with membrane 3024 sandwiched between retentate member 3022 and permeate/filtrate member 3020. Also, retentate member 3022, membrane member 3024, and permeate/filtrate member 3020 are coupled side-to-side with reservoir assembly 3050.
- Seen are two retentate ports 3028 (which couple the tangential flow channel 3002 in retentate member 3022 to the two retentate reservoirs (not shown), and one permeate/filtrate port 3026, which couples the tangential flow channel 3002 in permeate /filtrate member 3020 to the permeate/filtrate reservoir (not shown).
- tangential flow channel 3002 which is formed by the mating of retentate member 3022 and permeate/filtrate member 3020, with membrane 3024 sandwiched between and bifurcating tangential flow channel 3002.
- energy director 3091 which in this FIG.
- 3J has been used to ultrasonically weld or couple retentate member 3022 and permeate/filtrate member 3020, surrounding membrane 3024.
- Cover 3044 can be seen on top of reservoir assembly 3050, and gasket 3045 is disposed upon cover 3044. Gasket 3045 engages with and provides a fluid-tight seal and pneumatic connections with fluid transfer ports 3042 and pneumatic ports 3030, respectively.
- FIG. 3K depicts, on the left, an exploded view of the TFF module 3000 shown in FIG. 3J. Seen are components reservoir assembly 3050, a cover 3044 to be disposed on reservoir assembly 3050, a gasket 3045 to be disposed on cover 3044, retentate member 3022, membrane or filter 3024, and permeate/filtrate member 3020. Also seen is permeate/filtrate port 3026, which mates with permeate/filtrate port 3026 on permeate/filtrate reservoir 3054, as well as two retentate ports 3028, which mate with retentate ports 3028 on retentate reservoirs 3052 (where only one retentate reservoir 3052 can be seen clearly in this FIG. 3K).
- FIG. 3K depicts on the left the assembled TFF module 3000 showing length, height, and width dimensions.
- the assembled TFF device 3000 typically is from 50 to 175 mm in height, or from 75 to 150 mm in height, or from 90 to 120 mm in height; from 50 to 175 mm in length, or from 75 to 150 mm in length, or from 90 to 120 mm in length; and is from 30 to 90 mm in depth, or from 40 to 75 mm in depth, or from about 50 to 60 mm in depth.
- An exemplary TFF device is 110 mm in height, 120 mm in length, and 55 mm in depth.
- the TFF device or module depicted in FIGs. 3E - 3K can constantly measure cell culture growth, and in some aspects, cell culture growth is measured via optical density (OD) of the cell culture in one or both of the retentate reservoirs and/or in the flow channel of the TFF device.
- Optical density may be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5, 10, 15, 20, 3045, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or so on minutes.
- the TFF module can adjust growth parameters (temperature, aeration) to have the cells at a desired optical density at a desired time.
- FIG. 3E - 3K can constantly measure cell culture growth, and in some aspects, cell culture growth is measured via optical density (OD) of the cell culture in one or both of the retentate reservoirs and/or in the flow channel of the TFF device.
- Optical density may be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5,
- 3L is an exemplary pneumatic block diagram suitable for the TFF module depicted in FIGs. 3E - 3K.
- the pump is connected to two solenoid valves (SV5 and S V6) delivering positive pressure (P) or negative pressure (V).
- the two solenoid valves SV5 and SV6 couple the pump to the manifold, and two solenoid valves, SV1 and SV2, are connected to the reservoirs (RR1 and RR2).
- RR1 and RR2 reservoirs
- PV2 and PV2 proportional valve
- FM1 and FM2 flow meter
- Pressure Sensors 1 and 2 Pressure Sensors 1 and 2 positioned in between each of solenoid valves SV1 and SV2 connecting the pump to the system and the solenoid valves SV1 And SV2 to the reservoirs.
- the pressure sensors and prop valves work in concert in a feedback loop to maintain the required pressure.
- a cell concentration module comprising a hollow filter
- filters suitable for use in the present invention include membrane filters, ceramic filters and metal filters.
- the filter may be used in any shape; the filter may for example be cylindrical or essentially flat.
- the filter used is a membrane filter, preferably a hollow fiber filter.
- the term "hollow fiber” is meant a tubular membrane.
- the internal diameter of the tube is at least 0.1 mm, more preferably at least 0.5 mm, most preferably at least 0.75 mm and preferably the internal diameter of the tube is at most 10 mm, more preferably at most 6 mm, most preferably at most 1 mm.
- Filter modules comprising hollow fibers are commercially available from various companies, including G.E. Life Sciences (Marlborough, MA) and InnovaPrep (Drexel, MO).
- Specific examples of hollow fiber filter systems that can be used, modified or adapted for use in the present methods and systems include, but are not limited to, USPNs 9,738,918; 9,593,359; 9,574,977; 9,534,989; 9,446,354; 9,295,824; 8,956,880; 8,758,623; 8,726,744; 8,677,839; 8,677,840; 8,584,536; 8,584,535; and 8,110,112.
- FIGs. 4A - 4E depict variations on one embodiment of a module for introduction of editing machinery into cells.
- the introduction methods can be tailored depending on the cell type and nature of the machinery to be introduced (e.g., nucleic acids or proteins).
- the module is configured to transform mammalian cells.
- an editing cassette plasmid and nuclease can be delivered to the target cell by traditional mammalian cell transfection techniques. Examples include lipid- mediated transfection, Calcium Phosphate-mediated transfection, electroporation, cationic peptides, cationic polymers, or nucleofection.
- Proteins such as an RNA-directed nuclease can also be delivered to the cells using various mechanisms.
- an RNA-directed nuclease can be introduced to mammalian cells using shuttle vectors such as those described in USPNs 9,982,267 and 9,738,687, which are incorporated herein by reference for all purposes.
- FIG. 4A is a perspective view of six co-joined flowthrough electroporation devices 450.
- FIG. 4A depicts six flow-through electroporation units 450 arranged on a single substrate 456.
- Each of the six flow-through electroporation units 450 have wells 452 that define cell sample inlets and wells 454 that define cell sample outlets.
- the flow-through electroporation devices achieve high efficiency cell electroporation with low toxicity.
- the flow-through electroporation devices of the disclosure allow for particularly easy integration with robotic liquid handling instrumentation that is typically used in automated systems such as air displacement pipettors.
- robotic liquid handling instrumentation includes, but is not limited to, off-the-shelf automated liquid handling systems from Tec an (Mannedorf, Switzerland), Hamilton (Reno, NV), Beckman Coulter (Fort Collins, CO), etc.
- microfluidic electroporation using cell suspension volumes of less than approximately 10 ml and as low as 1 ⁇ l — allows more precise control over a transfection or transformation process and permits flexible integration with other cell processing tools compared to bench-scale electroporation devices.
- Microfluidic electroporation thus provides unique advantages for, e.g., single cell transformation, processing and analysis; multi-unit electroporation device configurations; and integrated, automatic, multi-module cell processing and analysis.
- the toxicity level of the transformation results in greater than 10% viable cells after electroporation, preferably greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or even 95% viable cells following transformation, depending on the cell type and the nucleic acids being introduced into the cells.
- the flow-through electroporation device described in relation to FIGs. 4A- 4D comprises a housing with an electroporation chamber, a first electrode and a second electrode configured to engage with an electric pulse generator, by which electrical contacts engage with the electrodes of the electroporation device.
- the electroporation devices are autoclavable and/or disposable, and may be packaged with reagents in a reagent cartridge.
- the electroporation device may be configured to electroporate cell sample volumes between 1 ⁇ l to 2 ml, 10 ⁇ l to 1 ml, 25 ⁇ l to 750 ⁇ 1, or 50 ⁇ l to 500 ⁇ l.
- FIG. 4B depicts a top view of a flow-through electroporation device 450 having an inlet 402 for introduction of cells and an exogenous reagent to be electroporated into the cells (“cell sample”) and an outlet 404 for the cell sample following electroporation. Electrodes 408 are introduced through electrode channels (not shown) in the device.
- FIG. 4C shows a cutaway view from the top of flow-through electroporation device 450, with the inlet 402, outlet 404, and electrodes 408 positioned with respect to a constriction in flow channel 406. A side cutaway view of the bottom portion of flow-through electroporation device 450 in FIG.
- Electrodes 408 in this embodiment are positioned in electrode channels 410 and perpendicular to flow channel 406 such that the cell sample flows from the inlet channel 412 through the flow channel 406 to the outlet channel 414, and in the process the cell sample flows into the electrode channels 410 to be in contact with electrodes 408.
- the inlet channel, outlet channel and electrode channels all originate from the top planar side of the device; however, the flow-through electroporation architecture depicted in FIGs.4B-4D is but one architecture useful with the reagent cartridges described herein. Additional electrode architectures are described, e.g., in USSNs. 16/147,120, filed 24 September 2018; 16/147,865, filed 30 September 2018; and 16/147,871, filed 30 September 2018.
- FIG. 5A depicts an exemplary combination reagent cartridge and electroporation device 500 (“cartridge”) that may be used in an automated multimodule cell processing instrument.
- Cartridge 500 comprises a body 502, and reagent receptacles or reservoirs 504. Additionally, cartridge 500 comprises a device for introduction of nucleic acids and/or proteins into the cells, e.g. an electroporation device 506 (an exemplary embodiment of which is described in detail in relation to FIGs. 4A- 4D.
- Cartridge 500 may be disposable, or may be configured to be reused. Preferably, cartridge 500 is disposable.
- Cartridge 500 may be made from any suitable material, including stainless steel, aluminum, or plastics including polyvinyl chloride, cyclic olefin copolymer (COC), polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymers of these and other polymers. If the cartridge is disposable, preferably it is made of plastic.
- plastics including polyvinyl chloride, cyclic olefin copolymer (COC), polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymers of these and other polymers.
- PEEK polyetheretheketone
- PMMA poly
- the material used to fabricate the cartridge is thermally-conductive, as in certain embodiments the cartridge 500 contacts a thermal device (not shown) that heats or cools reagents in the reagent receptacles or reservoirs 504.
- the thermal device is a Peltier device or thermoelectric cooler.
- Reagent receptacles or reservoirs 504 may be receptacles into which individual tubes of reagents are inserted as shown in FIG. 5A, receptacles into which one or more multiple co-joined tubes are inserted, or the reagent receptacles may hold the reagents without inserted tubes with the reagents dispensed directly into the receptacles or reservoirs.
- the receptacles in a reagent cartridge may be configured for any combination of tubes, cojoined tubes, and direct-fill of reagents.
- the reagent receptacles or reservoirs 504 of reagent cartridge 500 are configured to hold various size tubes, including, e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorf or microcentrifuge tubes.
- all receptacles may be configured to hold the same size tube, e.g., 5 ml tubes, and reservoir inserts may be used to accommodate smaller tubes in the reagent reservoir.
- the reagent reservoirs hold reagents without inserted tubes.
- the reagent cartridge may be part of a kit, where the reagent cartridge is pre-filled with reagents and the receptacles or reservoirs sealed with, e.g., foil, heat seal acrylic or the like and presented to a consumer where the reagent cartridge can then be used in an automated multi-module cell processing instrument.
- the reagents contained in the reagent cartridge will vary depending on work flow; that is, the reagents will vary depending on the processes to which the cells are subjected in the automated multi-module cell processing instrument.
- FIG. 5B depicts an exemplary matrix configuration 140 for the reagents contained in the reagent cartridges of FIG. 5A; where this matrix embodiment is a 4 X 4 reagent matrix.
- a user can locate the proper reagent for a given process. That is, reagents such as cell samples, enzymes, buffers, nucleic acid vectors, expression cassettes, reaction components (such as, e.g., MgCl 2 , dNTPs, isothermal nucleic acid assembly reagents, Gap Repair reagents, and the like), wash solutions, ethanol, and magnetic beads for nucleic acid purification and isolation, etc. are positioned in the matrix 540 at a known position.
- reagents such as cell samples, enzymes, buffers, nucleic acid vectors, expression cassettes, reaction components (such as, e.g., MgCl 2 , dNTPs, isothermal nucleic acid assembly reagents, Gap Repair reagents, and the like), wash solutions, ethanol, and magnetic beads for nucleic acid purification and isolation, etc.
- FIG. 5A is labeled to show where several reservoirs 504 correspond to matrix 540: See receptacles 510, 513, 521 and 525.
- matrices of the reagent cartridge and electroporation devices can be any configuration, such as, e.g., 2 X 2, 2 X 3, 2 X 4, 2X5, 2X 6, 3 X 3, 3 X 5, 4X6, 6X7, or any other configuration, including asymmetric configurations, or two or more different matrices depending on the reagents needed for the intended workflow.
- the matrix configuration is a 5 X 3 +1 matrix.
- the reagent cartridge comprises a script (not shown) readable by a processor (not shown) for dispensing the reagents via a liquid handling device (not shown) and controlling the electroporation device contained within reagent cartridge 500.
- the reagent cartridge 500 as one component in an automated multi-module cell processing instrument may comprise a script specifying two, three, four, five, ten or more processes performed by the automated multi-module cell processing instrument, or even specify all processes performed by the automated multi-module cell processing instrument.
- the reagent cartridge is disposable and is pre-packaged with reagents tailored to performing specific cell processing protocols, e.g., genome editing or protein production.
- reagent cartridges may be pre-packaged with reagents for genome editing and a script that specifies the process steps for performing genome editing in an automated multi-module cell processing instrument such as described in relation to FIGs. 1A - ID.
- the reagent cartridge may comprise a script to pipette electrocompetent cells from reservoir A2 (511), transfer the cells to the electroporation device 506, pipette a nucleic acid solution comprising an editing vector from reservoir C3 (520), transfer the nucleic acid solution to the electroporation device, initiate the electroporation process for a specified time, then move the porated cells to a reservoir D4 (525) in the reagent cassette or to another module such as the rotating growth vial (118 or 120 of FIG. 1A) in the automated multi-module cell processing instrument in FIG. 1 A.
- the reagent cartridge may comprise a script to pipette transfer of a nucleic acid solution comprising a vector from reservoir C3 (520), nucleic acid solution comprising editing oligonucleotide cassettes in reservoir C4 (521), and isothermal nucleic acid assembly reaction mix from A1 (510) to the isothermal nucleic acid assembly/desalting reservoir (414 of FIG. 4A).
- the script may also specify process steps performed by other modules in the automated multi-module cell processing instrument.
- the script may specify that the isothermal nucleic acid assembly/desalting module be heated to 50°C for 30 min to generate an assembled isothermal nucleic acid product; and desalting of the assembled isothermal nucleic acid product via magnetic bead-based nucleic acid purification involving a series of pipette transfers and mixing of magnetic beads in reservoir B2 (515), ethanol wash in reservoir B3 (516), and water in reservoir Cl (518) to the isothermal nucleic acid assembly/desalting reservoir (114 of FIG. 1A).
- the disclosure also includes automated multi-module cell editing instruments with an enrichment module that performs enrichment methods including those described herein to increase the overall editing efficiency in a population of cells, e.g., mammalian cells.
- the enrichment module can be designed to accommodate the particular enrichment method, and is preferably (but not required to be) connected to the remaining modules of the multi-module instrument, e.g. via an automated liquid handling system or other cell transfer device.
- the enrichment module can be a module used off- instrument, with the resulting enriched cell populations introduced back to the integrated instrument, or alternatively to a companion instrument that completes the editing and recovery cycle.
- the enrichment module acts independent from the automated multi-module instrument, but is included into the overall workflow.
- the work flow may require coordination of two or more processors responsible for different parts of the work flow.
- the enrichment module is in fluid communication with the automated multi-module instruments and integrated with a liquid handling system and controlled by a single processor.
- the enrichment is a positive enrichment module that enriches for cells that contain an introduced selection marker.
- the enrichment is a negative selection that depletes cells based on the lack of a selection marker or a characteristic that is absent due to the specific enrichment method used, e.g., antibiotic selection.
- the selection process can be performed computationally, and the expression of the selection marker monitored and used in future data analysis to determine the editing rate of a cell population.
- Certain selection methods that can be used with the methods of the present disclosure provides fluorescent or bioluminescent selection as a read out for properly- edited cells.
- the properly-edited cells can be sorted from non-edited or improperly- edited cells via methods such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), and modules for performing such selections can be incorporated into the automated multi-module cell processing instrument (see, e.g., 140 of FIG. 1 A).
- FACS fluorescence-activated cell sorting
- MCS magnetic-activated cell sorting
- FACS can isolate cells based on internal staining or intracellular protein expression, and allows for the purification of individual cells based on size, granularity and fluorescence.
- Cells in suspension are passed as a stream in droplets with each droplet containing a single cell of interest.
- the droplets are passed in front of a laser.
- An optical detection system detects cells of interest based on predetermined optical parameters (e.g., fluorescent or bioluminescent parameters).
- the instrument applies a charge to a droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate tubes or wells. Sorting parameters may be adjusted depending on the requirement of purity and yield.
- MACSTM (Miltenvi Biotec) is a method for separation of various cell populations depending on their surface antigens. This selection process relies on the co-introduction of cell-surface markers that are not otherwise present on the surface of cells to be edited.
- FIG. 6 illustrates an embodiment of a multi-module cell processing instrument.
- This embodiment depicts an exemplary system that performs recursive gene editing on a mammalian cell population.
- the cell processing instrument 600 may include a housing, a reservoir for storing cells to be transformed or transfected 604, and a cell growth and/or concentration module (comprising, e.g., a rotating growth vial) 608.
- the cells to be transformed are transferred from a reservoir to the cell growth module to be cultured until the cells hit a target OD. Once the cells hit the target OD, the growth module may cool or freeze the cells for later processing proceed to perform cell concentration where the cells are subjected to buffer exchange and rendered electrocompetent, and the volume of the cells may be reduced substantially.
- the multi- module cell processing instrument includes a reservoir for storing an editing vector pre- assembled with editing oligonucleotide cassettes 606.
- the pre-assembled editing vectors are transferred to the editing machinery introduction module 610, which already contains the cell culture grown to a target OD.
- the instrument may comprise a reservoir 602 for storing an engine vector comprising the coding sequence for the nucleic acid-guided nuclease.
- the engine vectors may be transferred to the editing machinery introduction module 610 and transformed at the same time the editing vectors are transformed, or the engine vectors may be transformed into the cells before or after the editing vectors have been transformed into the cells.
- the nucleic acids are, e.g., electroporated into the cells.
- the cells are transferred into an optional recovery module (not shown), where the cells recover briefly post-transformation.
- the cells may be transferred to a storage module (also not shown), where the cells can be stored at, e.g., 4°C for later processing.
- selection may be optionally performed in a separate module between the editing machinery introduction module and the editing module, or selection may be performed in the editing module. Selection in this instance refers to selecting for cells that have been properly transformed with vectors that comprise selection markers, thus assuring that the cells are likely to have received vectors for both nucleic acid-guided nuclease editing and for reporting proper edits.
- the cells may optionally be diluted and transferred to an editing module 612. Conditions are then provided such that editing takes place.
- the editing components e.g., one or more of the nucleic acid-guided nuclease, gRNA or donor DNA
- conditions are provided to activate the inducible promoters.
- cells are selected in an enrichment module 614 where the cells are selected, e.g., sorted using FACS or MACSTM. Cells expressing the selection marker are separated in the enrichment module from cells that do not express the expression marker, and optionally prepared for another round of editing.
- the multi- module cell processing instrument is controlled by a processor 616 configured to operate the instrument based on user input, as directed by one or more scripts, or as a combination of user input or a script.
- the processor 616 may control the timing, duration, temperature, and operations of the various modules of the instrument 600 and the dispensing of reagents from the reagent cartridge.
- the processor may be programmed with standard protocol parameters from which a user may select, a user may specify one or more parameters manually or one or more scripts associated with the reagent cartridge may specify one or more operations and/or reaction parameters.
- the processor may notify the user (e.g., via an application to a smart phone or other device) that the cells have reached a target OD, been rendered competent and concentrated, and/or update the user as to the progress of the cells in the various modules in the multi-module instrument.
- the process described may be recursive and multiplexed; that is, cells may go through the workflow described in relation to FIG. 6, then the resulting edited culture may go through another (or several or many) rounds of additional editing (e.g., recursive editing) with different editing vectors.
- the cells from round 1 of editing may be diluted and an aliquot of the edited cells edited by editing vector A may be combined with editing vector B, an aliquot of the edited cells edited by editing vector A may be combined with editing vector C, an aliquot of the edited cells edited by editing vector A may be combined with editing vector D, and so on for a second round of editing.
- an aliquot of each of the double-edited cells may be subjected to a third round of editing, where, e.g., aliquots of each of the AB-, AC-, AD- edited cells are combined with additional editing vectors, such as editing vectors X, Y, and Z.
- double-edited cells AB may be combined with and edited by vectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, and ABZ
- double-edited cells AC may be combined with and edited by vectors X, Y, and Z to produce triple- edited cells ACX, ACY, and ACZ
- double-edited cells AD may be combined with and edited by vectors X, Y, and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on.
- many permutations and combinations of edits can be executed, leading to very diverse cell populations and cell libraries.
- “cure” is a process in which one or more vectors used in the prior round of editing is eliminated from the transformed cells.
- Curing can be accomplished by, e.g., cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector (or single, combined engine/editing vector) nonfunctional; diluting the vectors) in the cell population via cell growth (that is, the more growth cycles the cells go through, the fewer daughter cells will retain the editing or engine vector(s)), or by, e.g., utilizing a heat-sensitive origin of replication on the editing or engine vector (or combined engine + editing vector).
- the conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing and/or engine vector.
- nucleic acid-directed nuclease editing methods with selection procedures - either computational or physical, as described further herein - results in a significant increase in editing efficiency in comparison to the editing methods without such selection methods.
- the editing workflow consists of the use of a nuclease (e.g., an RNA-directed nuclease such as cas-9, cpf-1, MAD7, and the like) with one or more selection events to increase editing rates in cells, including increasing the editing rates in mammalian cells.
- a nuclease e.g., an RNA-directed nuclease such as cas-9, cpf-1, MAD7, and the like
- FIG. 7 shows an exemplary workflow in which editing machinery and the coding sequences for an RNA-directed nuclease are delivered to cells in two separate vectors.
- the workflow includes design of gRNAs targeting the region of a genome to be edited, covalently attached to a homology arm containing one or more intended edits 702.
- the edits include an edit to render the target site resistant to further nuclease cleavage, e.g., a mutation in a PAM site and/or spacer region.
- These gRNA-HA constructs are introduced to editing vectors 704 that includes a promoter for expression of the nucleic acids and optionally includes a barcode or other mechanism to track a specific edit.
- the promoter used to drive the editing machinery is inducible.
- the coding sequences for an RNA-directed nuclease are introduced into a second set of vectors 708 to create engine vectors.
- the engine vectors have the coding sequences of the nuclease under a separate promoter from the editing vectors.
- the separate promoter of the engine vectors may be the same or different than the promoter used for the editing vector, and optionally is inducible.
- the engine vectors and editing vectors are introduced to cells 710, e.g., using transformation, transfection, or other mechanisms that will be apparent to one of skill in the art upon reading the present disclosure. The cells are then provided with conditions for editing the cells 712, and allowed to edit.
- the cells are selected 714 for the cells enriched for editing using techniques such as those described herein.
- Such techniques could use computational means of selection for further analysis of the edited cell population as well as physical selection using negative selection and/or positive selection, such as selection of a selection marker e.g., a cell-surface marker that can serve as a handle for physical enrichment of the putatively edited cells.
- FIG. 8 shows an exemplary workflow using a single vector system to introduce both the editing nucleic acids and the coding sequences for a nuclease to a cell population to be edited.
- the workflow includes design of gRNAs targeting the region of a genome to be edited, covalently attached to a homology arm containing one or more intended edits 802.
- the edits include an edit to render the target site resistant to further nuclease cleavage, e.g., a mutation in a PAM site and/or spacer region.
- gRNA-HA constructs and coding sequences for a nuclease are introduced 804 to the same vectors to create a single vector that includes one or more promoters for expression of the nucleic acids and the nuclease.
- the single vector optionally includes a barcode or other mechanism to track a specific edit.
- the vector may contain a single promoter for expression of both the gRNA-HA constructs and coding sequences for a nuclease, or the gRNA-HA constructs and coding sequences for a nuclease may be under the control of different promoters in the same vector.
- the promoter or promoters used to drive the editing machinery and/or the coding for the nuclease are inducible.
- the vectors are introduced to cells 810, e.g., using transformation, transfection, or other mechanisms that will be apparent to one of skill in the art upon reading the present disclosure.
- the cells are then provided with conditions for editing the cells 812, and allowed to edit.
- the cells are selected 814 for the cells enriched for editing using techniques such as those described herein. Such techniques could use computational means of selection for further analysis of the edited cell population as well as physical selection using negative selection and/or positive selection, such as selection of a selection marker e.g., a cell-surface marker that can serve as a handle for physical enrichment of the putatively edited cells.
- FGS. 9 shows an exemplary workflow in which editing machinery and the coding sequences for an RNA-directed nuclease are delivered to cells in two separate vectors.
- the workflow includes design of gRNAs targeting the region of a genome to be edited, covalently attached to a homology arm containing one or more intended edits 902.
- the edits include an edit to render the target site resistant to further nuclease cleavage, e.g., a mutation in a PAM site and/or spacer region.
- These gRNA-HA constructs are introduced to editing vectors 904 that includes a promoter for expression of the nucleic acids and optionally includes a barcode or other mechanism to track a specific edit.
- the promoter used to drive the editing machinery is inducible.
- the coding sequences for a fusion vector of an RNA-directed nuclease ⁇ e.g., cas-9, cpf-1, MAD7) and an enzyme region with desired functionality ⁇ e.g., reverse transcriptase activity
- the engine vectors have the coding sequences of the nuclease under a separate promoter from the editing vectors.
- the separate promoter of the engine vectors may be the same or different that the promoter used for the editing vector, and optionally is inducible.
- the engine vectors and editing vectors are introduced to cells 910, e.g., using transformation, transfection, or other mechanisms that will be apparent to one of skill in the art upon reading the present disclosure.
- the cells are then provided with conditions for editing the cells 912, and allowed to edit.
- the cells are selected 914 for the cells enriched for editing using techniques such as those described herein.
- Such techniques could use computational means of selection for further analysis of the edited cell population as well as physical selection using negative selection and/or positive selection, such as selection of a selection marker e.g., a cell-surface marker that can serve as a handle for physical enrichment of the putatively edited cells.
- FIG. 10 shows an exemplary workflow using a single vector system to introduce both the editing nucleic acids and the coding sequences for a nuclease to a cell population to be edited.
- the workflow includes design of gRNAs targeting the region of a genome to be edited, covalently attached to a homology arm containing one or more intended edits 1002.
- the edits include an edit to render the target site resistant to further nuclease cleavage, e.g., a mutation in a PAM site and/or spacer region.
- gRNA-HA constructs and coding sequences for a fusion vector of an RNA-directed nuclease e.g., cas-9, cpf-1, MAD7
- an enzyme region with desired functionality e.g., reverse transcriptase activity
- the single vector optionally includes a barcode or other mechanism to track a specific edit.
- the vector may contain a single promoter for expression of both the gRNA-HA constructs and coding sequences for the fusion protein, or the gRNA-HA constructs and coding sequences for the fusion protein may be under the control of different promoters in the same vector.
- the promoter or promoters used to drive the editing machinery and/or the coding for the fusion protein are inducible.
- the vectors are introduced to cells 1010, e.g., using transformation, transfection, or other mechanisms that will be apparent to one of skill in the art upon reading the present disclosure.
- the cells are then provided with conditions for editing the cells 812, and allowed to edit.
- the cells are selected 1014 for the cells enriched for editing using techniques such as those described herein. Such techniques could use computational means of selection for further analysis of the edited cell population as well as physical selection using negative selection and/or positive selection, such as selection of a selection marker e.g., a cell-surface marker that can serve as a handle for physical enrichment of the putatively edited cells.
- a selection marker e.g., a cell-surface marker that can serve as a handle for physical enrichment of the putatively edited cells.
- the steps 1010-1014 (or in some cases, 1012-1014 if sufficient editing and/or engine vectors are present in the cell population and do not need to be added again) can optionally be repeated 1016 to increase editing efficiency of the cell population.
- the present disclosure provides editing methods, modules, instruments, and automated multi-module cell editing instruments for creating a library of cells that vary the expression, levels and/or activity of RNAs and/or proteins of interest in various cell types using various nickase-based editing strategies, including CREATE fusion, as described herein in more detail.
- the disclosure is intended to cover edited cell libraries created by the automated editing methods, automated multi-module cell editing instruments of the disclosure.
- These cell libraries may have different targeted edits, including but not limited to gene knockouts, gene knock-ins, insertions, deletions, single nucleotide edits, short tandem repeat edits, frameshifts, triplet codon expansion, and the like in cells of various organisms. These edits can be directed to coding or non-coding regions of the genome, and are preferably rationally designed.
- the present disclosure provides automated editing methods, automated multi-module cell editing instruments for creating a library of cells that vary DNA-linked processes.
- the cell library may include individual cells having edits in DNA binding sites to interfere with DNA binding of regulatory elements that modulate expression of selected genes.
- cell libraries may include edits in genomic DNA that impact on cellular processes such as heterochromatin formation, switch-class recombination and VDJ recombination.
- the cell libraries are created using multiplexed, nickase- directed editing of individual cells within a cell population, with multiple cells within a cell population are edited in a single round of editing, i.e., multiple changes within the cells of the cell library are in a single automated operation.
- the libraries that can be created in a single multiplexed automated operation can comprise as many as 500 cells with intended edits, which may be the same introduced edit in the cells or two or more discrete edits in different cells.
- the libraries can also include one or more intended edits (the same or different) in 1000 edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells, 10,000,000 edited cells or more.
- intended edits the same or different
- the cell libraries are created using nickase-directed recursive editing of individual cells within a cell population, with edits being added to the individual cells in two or more rounds of editing.
- the use of recursive editing results in the amalgamation of two or more edits targeting two or more sites in the genome in individual cells of the library.
- the libraries that can be created in a single multiplexed automated operation can comprise as many as 500 cells with intended edits, which may be the same introduced edit in the cells or two or more discrete edits in different cells.
- the libraries can also include one or more intended edits (the same or different) in 1000 edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells, 10,000,000 edited cells or more.,
- recursive editing can be used to first create a cell phenotype, and then later rounds of editing used to reverse the phenotype and/or accelerate other cell properties.
- the cell library comprises edits for the creation of unnatural amino acids in a cell.
- the disclosure provides edited cell libraries having edits in one or more regulatory elements created using the disclosed editing methods, automated multi-module cell editing instruments of the disclosure.
- regulatory element refers to nucleic acid molecules that can influence the transcription and/or translation of an operably linked coding sequence in a particular environment and/or context. This term is intended to include all elements that promote or regulate transcription, and RNA stability including promoters, core elements required for basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements (see, e.g., Lewin, "Genes V” (Oxford University Press, Oxford) pages 847-873). Exemplary regulatory elements in prokaryotes include, but are not limited to, promoters, operator sequences and a ribosome binding sites. Regulatory elements that are used in eukaryotic cells may include, but are not limited to, promoters, enhancers, insulators, splicing signals and polyadenylation signals.
- the edited cell library includes rationally designed edits that are designed based on predictions of protein structure, expression and/or activity in a particular cell type.
- rational design may be based on a system-wide biophysical model of genome editing with a particular nuclease and gene regulation to predict how different editing parameters including nuclease expression and/or binding, growth conditions, and other experimental conditions collectively control the dynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS Comput Biol.,
- the present disclosure provides the creation of a library of edited cells with various rationally designed regulatory sequences created using the nickase methods of the disclosure, including automated methods using the disclosed instrument.
- the edited cell library can include prokaryotic cell populations created using set of constitutive and/or inducible promoters, enhancer sequences, operator sequences and/or ribosome binding sites.
- the edited cell library can include eukaryotic sequences created using a set of constitutive and/or inducible promoters, enhancer sequences, operator sequences, and/or different Kozak sequences for expression of proteins of interest.
- the disclosure provides cell libraries including cells with rationally designed edits comprising one or more classes of edits in sequences of interest across the genome of an organism.
- the disclosure provides cell libraries including cells with rationally designed edits comprising one or more classes of edits in sequences of interest across a subset of the genome.
- the cell library may include cells with rationally designed edits comprising one or more classes of edits in sequences of interest across the exome, e.g., every or most open reading frames of the genome.
- the cell library may include cells with rationally designed edits comprising one or more classes of edits in sequences of interest across the kinome.
- the cell library may include cells with rationally designed edits comprising one or more classes of edits in sequences of interest across the secretome.
- the cell library may include cells with rationally designed edits created to analyze various isoforms of proteins encoded within the exome, and the cell libraries can be designed to control expression of one or more specific isoforms, e.g., for transcriptome analysis.
- the cell libraries may comprise edits using randomized sequences, e.g., randomized promoter sequences, to reduce similarity between expression of one or more proteins in individual cells within the library.
- the promoters in the cell library can be constitutive, inducible or both to enable strong and/or titratable expression.
- the present disclosure provides nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for creating a library of cells comprising edits to identify optimum expression of a selected gene target.
- production of biochemicals through metabolic engineering often requires the expression of pathway enzymes, and the best production yields are not always achieved by the highest amount of the target pathway enzymes in the cell, but rather by fine- tuning of the expression levels of the individual enzymes and related regulatory proteins and/or pathways.
- expression levels of heterologous proteins sometimes can be experimentally adjusted for optimal yields.
- RNAp recruitment and transcriptional complex formation This process, known as transcriptional interference, is particularly relevant in lower eukaryotes, as they often have closely spaced genes.
- the present disclosure provides methods for optimizing cellular gene transcription.
- Gene transcription is the result of several distinct biological phenomena, including transcriptional initiation (RNAp recruitment and transcriptional complex formation), elongation (strand synthesis/extension), and transcriptional termination (RNAp detachment and termination).
- Cell libraries can be created using the nickase-based editing methods, modules, instruments and systems employing site-directed mutagenesis, i.e., when the amino acid sequence of a protein or other genomic feature may be altered by deliberately and precisely by mutating the protein or genomic feature.
- site-directed mutagenesis can be useful for various purposes, e.g., for determining protein function within cells, the identification of enzymatic active sites within cells, and the design of novel proteins.
- site-directed mutagenesis can be used in a multiplexed fashion to exchange a single amino acid in the sequence of a protein for another amino acid with different chemical properties. This allows one to determine the effect of a rationally designed or randomly generated mutation genes in individual cells within a cell population.
- edits can be made to individual cells within a cell library to substitute amino acids in binding sites, such as substitution of one or more amino acids in a protein binding site for interaction within a protein complex or substitution of one or more amino acids in enzymatic pockets that can accommodate a cofactor or ligand.
- This class of edits allows the creation of specific manipulations to a protein to measure certain properties of one or more proteins, including interaction with other cofactors, ligands, etc. within a protein complex.
- eQTLs are a locus that explains a fraction of the genetic variance of a gene expression phenotype.
- the libraries of the invention would be useful to evaluate and link eQTLs to actual diseased states.
- the edits introduced into the cell libraries of the disclosure may be created using rational design based on known or predicted structures of proteins. See, e.g., Chronopoulou EG and Labrou, Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011).
- site-directed mutagenesis can provide individual cells within a library with one or more site-directed edits, and preferably two or more site-directed edits (e.g., combinatorial edits) within a cell population.
- cell libraries of the disclosure are created using site-directed codon mutation “scanning” of all or substantially all of the codons in the coding region of a gene.
- alanine scanning can be used to determine the contribution of a specific residue to the stability or function of given protein. See, e.g., Lefberg, et al., Nucleic Acids Research, Volume 25(2):447-448 (1997). Alanine is often used in this codon scanning technique because of its non-bulky, chemically inert, methyl functional group that can mimic the secondary structure preferences that many of the other amino acids possess. Codon scanning can also be used to determine whether the side chain of a specific residue plays a significant role in cell function and/or activity. Sometimes other amino acids such as valine or leucine can be used in the creation of codon scanning cell libraries if conservation of the size of mutated residues is needed.
- cell libraries can be created using the nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments of the disclosure to determine the active site of a protein such as an enzyme or hormone, and to elucidate the mechanism of action of one or more of these proteins in a cell library.
- Site-directed mutagenesis associated with molecular modeling studies can be used to discover the active site structure of an enzyme and consequently its mechanism of action. Analysis of these cell libraries can provide an understanding of the role exerted by specific amino acid residues at the active sites of proteins, in the contacts between subunits of protein complexes, on intracellular trafficking and protein stability/half-life in various genetic backgrounds.
- the cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments are saturation mutagenesis libraries, in which a single codon or set of codons is randomized to produce all possible amino acids at the position of a particular gene or genes of interest.
- These cell libraries can be particularly useful to generate variants, e.g., for directed evolution. See, e.g., Chica, et al., Current Opinion in Biotechnology 16 (4): 378-384 (2005); and Shivange, Current Opinion in Chemical Biology, 13 (1): 19-25.
- edits comprising different degenerate codons can be used to encode sets of amino acids in the individual cells in the libraries. Because some amino acids are encoded by more codons than others, the exact ratio of amino acids cannot be equal. In certain aspects, more restricted degenerate codons are used. ' ⁇ ' and ’NNS’ have the benefit of encoding all 20 amino acids, but still encode a stop codon 3% of the time. Alternative codons such as ’NDT, ⁇ ’ avoid stop codons entirely, and encode a minimal set of amino acids that still encompass all the main biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small). [00234] In specific aspects, the non-redundant saturation mutagenesis, in which the most commonly used codon for a particular organism, is used in the saturation mutagenesis editing process.
- One mechanism for analyzing and/or optimizing expression of one or more genes of interest is through the creation of a “promoter swap” cell library, in which the cells comprise genetic edits that have specific promoters linked to one or more genes of interest.
- the cell libraries created nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments may be promoter swap cell libraries, which can be used, e.g., to increase or decrease expression of a gene of interest to optimize a metabolic or genetic pathway.
- the promoter swap cell library can be used to identify an increase or reduction in the expression of a gene that affects cell vitality or viability, e.g., a gene encoding a protein that impacts on the growth rate or overall health of the cells.
- the promoter swap cell library can be used to create cells having dependencies and logic between the promoters to create synthetic gene networks.
- the promoter swaps can be used to control cell to cell communication between cells of both homogeneous and heterogeneous (complex tissues) populations in nature.
- the cell libraries can utilize any given number of promoters that have been grouped together based upon exhibition of a range of expression strengths and any given number of target genes.
- the ladder of promoter sequences vary expression of at least one locus under at least one condition. This ladder is then systematically applied to a group of genes in the organism using the automated editing methods, automated multi-module cell editing instruments of the disclosure.
- the cell library formed using nickase-based editing methods include individual cells that are representative of a given promoter operably linked to one or more target genes of interest in an otherwise identical genetic background. Examples of non- automated editing strategies that can be modified to utilize the automated systems can be found, e.g., in US Pat. No. 9,988,624.
- the promoter swap cell library is produced by editing a set of target genes to be operably linked to a pre-selected set of promoters that act as a “promoter ladder” for expression of the genes of interest.
- the cells are edited so that one or more individual genes of interest are edited to be operably linked with the different promoters in the promoter ladder.
- an endogenous promoter does not exist, its sequence is unknown, or it has been previously changed in some manner, the individual promoters of the promoter ladder can be inserted in front of the genes of interest.
- These produced cell libraries have individual cells with an individual promoter of the ladder operably linked to one or more target genes in an otherwise identical genetic context.
- the promoters are generally selected to result in variable expression across different loci, and may include inducible promoters, constitutive promoters, or both.
- the set of target genes edited using the promoter ladder can include all or most open reading frames (ORFs) in a genome, or a selected subset of the genome, e.g., the ORFs of the kinome or a secretome.
- the target genes can include coding regions for various isoforms of the genes, and the cell libraries can be designed to expression of one or more specific isoforms, e.g., for transcriptome analysis using various promoters.
- the set of target genes can also be genes known or suspected to be involved in a particular cellular pathway, e.g. a regulatory pathway or signaling pathway.
- the set of target genes can be ORFs related to function, by relation to previously demonstrated beneficial edits (previous promoter swaps or previous SNP swaps), by algorithmic selection based on epistatic interactions between previously generated edits, other selection criteria based on hypotheses regarding beneficial ORF to target, or through random selection.
- the target genes can comprise non-protein coding genes, including non-coding RNAs.
- Editing of other functional genetic elements can also be used to systematically vary the expression level of a set of target genes, and can be introduced using the methods, automated multi-module cell editing instruments of the disclosure.
- a population of cells is edited using a ladder of enhancer sequences, either alone or in combination with selected promoters or a promoter ladder, to create a cell library having various edits in these enhancer elements.
- a population of cells is edited using a ladder of ribosome binding sequences, either alone or in combination with selected promoters or a promoter ladder, to create a cell library having various edits in these ribosome binding sequences.
- a population of cells is edited to allow the attachment of various mRNA and/or protein stabilizing or destabilizing sequences to the 5’ or 3’ end, or at any other location, of a transcript or protein.
- a population of cells of a previously established cell line may be edited using the automated editing methods, modules, instruments, and systems of the disclosure to create a cell library to improve the function, health and/or viability of the cells.
- many industrial strains currently used for large scale manufacturing have been developed using random mutagenesis processes iteratively over a period of many years, sometimes decades. Unwanted neutral and detrimental mutations were introduced into strains along with beneficial changes, and over time this resulted in strains with deficiencies in overall robustness and key traits such as growth rates.
- mammalian cell lines continue to mutate through the passage of the cells over periods of time, and likewise these cell lines can become unstable and acquire traits that are undesirable.
- the automated editing methods, automated multimodule cell editing instruments of the disclosure can use editing strategies such as SNP and/or STR swapping, indel creation, or other techniques to remove or change the undesirable genome sequences and/or introducing new genome sequences to address the deficiencies while retaining the desirable properties of the cells.
- editing strategies such as SNP and/or STR swapping, indel creation, or other techniques to remove or change the undesirable genome sequences and/or introducing new genome sequences to address the deficiencies while retaining the desirable properties of the cells.
- the editing in the individual cells in the edited cell library can incorporate the inclusion of “landing pads” in an ectopic site in the enome (e.g., a CarT locus) to optimize expression, stability and/or control.
- each library produced having individual cells comprising one or more edits is cultured and analyzed under one or more criteria (e.g., production of a chemical or product of interest).
- the cells possessing the specific criteria are then associated, or correlated, with one or more particular edits in the cell.
- criteria e.g., production of a chemical or product of interest.
- the identification of multiple edits associated with particular criteria or enhanced functionality/robustness may lead to cells with highly desirable characteristics.
- the cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments may be “knock-out” (KO) or “knock-in” (KI) edits of various genes of interest.
- the disclosure is intended to cover edited cell libraries created by the nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments that have one or more mutations that remove or reduce the expression of selected genes of interest to interrogate the effect of these edits on gene function in individual cells within the cell library.
- the cell libraries can be created using targeted gene KO (e.g., via insertion/deletion) or KOs (e.g., via homologous directed repair). For example, double strand breaks are often repaired via the non-homologous end joining DNA repair pathway. The repair is known to be error prone, and thus insertions and deletions may be introduced that can disrupt gene function.
- the edits are rationally designed to specifically affect the genes of interest, and individual cells can be created having a KI or KI of one or more locus of interest. Cells having a KO or KI of two or more loci of interest can be created using automated recursive editing of the disclosure.
- the KO or KI cell libraries are created using simultaneous multiplexed editing of cells within a cell population, and multiple cells within a cell population are edited in a single round of editing, i.e., multiple changes within the cells of the cell library are in a single automated operation.
- the cell libraries are created using recursive editing of individual cells within a cell population, and results in the amalgamation of multiple edits of two or more sites in the genome into single cells.
- cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments may be produced for systematically introducing or substituting single nucleotide polymorphisms (“SNPs”) into the genomes of the individual cells to create a “SNP swap” cell library,
- SNPs single nucleotide polymorphisms
- the SNP swapping methods of the present disclosure include both the addition of beneficial SNPs, and removing detrimental and/or neutral SNPs.
- the SNP swaps may target coding sequences, non-coding sequences, or both.
- a cell library is created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for systematically introducing or substituting short tandem repeats (“STR”) into the genomes of the individual cells to create an “STR swap” cell library.
- the STR swapping methods of the present disclosure include both the addition of beneficial STRs, and removing detrimental and/or neutral STRs.
- the STR swaps may target coding sequences, noncoding sequences, or both.
- the SNP and/or STR swapping used to create the cell library is multiplexed, and multiple cells within a cell population are edited in a single round of editing, i.e., multiple changes within the cells of the cell library are in a single automated operation.
- the SNP and/or STR swapping used to create the cell library is recursive, and results in the amalgamation of multiple beneficial sequences and/or the removal of detrimental sequences into single cells. Multiple changes can be either a specific set of defined changes or a partly randomized, combinatorial library of mutations, Removal of detrimental mutations and consolidation of beneficial mutations can provide immediate improvements in various cellular processes.
- SNP swapping overcomes fundamental limitations of random mutagenesis approaches as it is not a random approach, but rather the systematic introduction or removal of individual mutations across cells.
- RNA splicing is the process during which introns are excised and exons are spliced together to create the mRNA that is translated into a protein.
- the precise recognition of splicing signals by cellular machinery is critical to this process.
- cell libraries of the disclosure include a cell library created using nickase- based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for systematically introducing changes to known and/or predicted splice donor and/or acceptor sites in various loci to create a library of splice site variants of various genes.
- Such editing can help to elucidate the biological relevance of various isoforms of genes in a cellular context.
- Sequences for rational design of splicing sites of various coding regions can be predicted using analysis techniques such as those found in Nalla and Rogan, Hum Mutat, 25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009); Desmet, et el., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMC Bioinformatics, 12(suppl 4):S2 (2011).
- the present disclosure provides for the creation of cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for swapping start and stop codon variants throughout the genome of an organism or for a selected subset of coding regions in the genome, e.g., the kinome or secretome.
- individual cells will have one or more start or stop codons repladng the native start or stop codon for one or more gene of interest.
- typical start codons used by eukaryotes are ATG (AUG) and prokaryotes use ATG (AUG) the most, followed by GTG (GUG) and TTG (UUG).
- the cell library may include individual cells having substitutions for the native start codons for one or more genes of interest.
- the present disclosure provides for creation of a cell library by replacing ATG start codons with TTG in front of selected genes of interest.
- the present disclosure provides for automated creation of a cell library by replacing ATG start codons with GTG.
- the present disclosure provides for automated creation of a cell library by replacing GTG start codons with ATG.
- the present disclosure provides for automated creation of a cell library by replacing GTG start codons with TTG. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TTG start codons with ATG. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TTG start codons with GTG.
- typical stop codons for 5. cerevisiae and mammals are TAA (UAA) and TGA (UGA), respectively.
- the typical stop codon for monocotyledonous plants is TGA (UGA)
- insects and E. coli commonly use TAA (UAA) as the stop codon (Dalphin. et al., Nucl. Acids Res., 24: 216-218 (1996)).
- the cell library may include individual cells having substitutions for the native stop codons for one or more genes of interest.
- the present disclosure provides for automated creation of a cell library by replacing TAA stop codons with TAG. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TAA stop codons with TGA. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TGA stop codons with TAA. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TGA stop codons with TAG. In other aspects, the present disclosure provides for automated creation of a cell library by replacing TAG stop codons with TAA. In other aspects, the present invention teaches automated creation of a cell library by replacing TAG stop codons with TGA.
- cell libraries of the disclosure include a terminator swap cell library created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments. Terminator swap cell libraries can be used, e.g., to affect mRNA stability by releasing transcripts from sites of synthesis.
- the terminator swap cell library can be used to identify an increase or reduction in the efficiency of transcriptional termination and thus accumulation of unspliced pre-mRNA (e.g., West and Proudfoot, Mol Cell.; 33(3-9); 354-364 (2009) and/or 3’ end processing (e.g., West, et al., Mol Cell. 29(5):600-10 (2008)).
- unspliced pre-mRNA e.g., West and Proudfoot, Mol Cell.; 33(3-9); 354-364 (2009) and/or 3’ end processing (e.g., West, et al., Mol Cell. 29(5):600-10 (2008).
- the edits may edit a combination of edits to multiple terminators that are associated with a gene. Additional amino acids may also be added to the ends of proteins to determine the effect on the protein length on terminators.
- the cell libraries can utilize any given number of edits of terminators that have been selected for the terminator ladder based upon exhibition of a range of activity and any given number of target genes.
- the ladder of terminator sequences vary expression of at least one locus under at least one condition. This ladder is then systematically applied to a group of genes in the organism using the automated editing methods, modules, instruments and systems of the disclosure.
- the present disclosure provides for the creation of cell libraries using the automated editing methods, modules, instruments and systems of disclosure, where the libraries are created to edit terminator signals in one or more regions in the genome in the individual cells of the library. Transcriptional termination in eukaryotes operates through terminator signals that are recognized by protein factors associated with the RNA polymerase ⁇ .
- the cell library may contain individual eukaryotic cells with edits in genes encoding polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) and or gene encoding proteins recruited by CPSF and CstF factors to termination sites.
- CPSF polyadenylation specificity factor
- CstF cleavage stimulation factor
- the cell library may contain individual prokaryotic cells with edits in genes encoding proteins that affect the binding, efficiency and/or activity of these termination pathways.
- the present disclosure provides methods of selecting termination sequences ("terminators") with optimal properties.
- the present disclosure teaches provides methods for introducing and/or editing one or more terminators and/or generating variants of one or more terminators within a host cell, which exhibit a range of activity.
- a particular combination of terminators can be grouped together as a terminator ladder, and cell libraries of the disclosure include individual cells that are representative of terminators operably linked to one or more target genes of interest in an otherwise identical genetic background. Examples of non- automated editing strategies that can be modified to utilize the automated instruments can be found, e.g., in US Pat. No.
- the terminator swap cell library is produced by editing a set of target genes to be operably linked to a pre-selected set of terminators that act as a “terminator ladder” for expression of the genes of interest.
- the cells are edited so that the endogenous promoter is operably linked to the individual genes of interest are edited with the different promoters in the promoter ladder.
- the individual promoters of the promoter ladder can be inserted in front of the genes of interest.
- the terminator ladder can be used to more generally affect termination of all or most ORFs in a genome, or a selected subset of the genome, e.g., the ORFs of a kinome or a secretome.
- the set of target genes can also be genes known or suspected to be involved in a particular cellular pathway, e.g. a regulatory pathway or signaling pathway.
- the set of target genes can be ORFs related to function, by relation to previously demonstrated beneficial edits (previous promoter swaps or previous SNP swaps), by algorithmic selection based on epistatic interactions between previously generated edits, other selection criteria based on hypotheses regarding beneficial ORF to target, or through random selection.
- the target genes can comprise non-protein coding genes, including non-coding RNAs.
- EXAMPLES [00264] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
- the cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation was performed for 30 seconds.
- the parameters for the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1; polarity, +.
- the parameters for the transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/-.
- the cells were transferred to a recovery module (another growth module), and allowed to recover in SOC medium containing chloramphenicol. Carbenicillin was added to the medium after 1 hour, and the cells were allowed to recover for another 2 hours. After recovery, the cells were held at 4°C until recovered by the user.
- the result of the automated processing was that approximately l.OE -03 total cells were transformed (comparable to conventional benchtop results), and the editing efficiency was 83.5%.
- the lacZ_172 edit in the white colonies was confirmed by sequencing of the edited region of the genome of the cells. Further, steps of the automated cell processing were observed remotely by webcam and text messages were sent to update the status of the automated processing procedure.
- the first assembled editing vector and the recombineering-ready electrocompetent E.Coli cells were transferred into a editing machinery introduction module for electroporation.
- the cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation was performed for 30 seconds.
- the parameters for the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1; polarity, +.
- the parameters for the transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/-.
- the cells were transferred to a recovery module (another growth module) allowed to recover in SOC medium containing chloramphenicol.
- Carbenicillin was added to the medium after 1 hour, and the cells were grown for another 2 hours. The cells were then transferred to a centrifuge module and a media exchange was then performed. Cells were resuspended in TB containing chloramphenicol and caibenicillin where the cells were grown to OD600 of 2.7, then concentrated and rendered electrocompetent.
- a second editing vector was prepared in an isothermal nucleic acid assembly module. The second editing vector comprised a kanamycin resistance gene, and the editing cassette comprised a galK Y145* edit. If successful, the galK Y145* edit confers on the cells the ability to uptake and metabolize galactose.
- the edit generated by the galK Y154* cassette introduces a stop codon at the 154th amino acid reside, changing the tyrosine amino acid to a stop codon.
- This edit makes the galK gene product non-functional and inhibits the cells from being able to metabolize galactose.
- the second editing vector product was desalted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer.
- the assembled second editing vector and the electrocompetent cells were transferred into a editing machinery introduction module for electroporation, using the same parameters as detailed above, Following electroporation, the cells were transferred to a recovery module (another growth module), allowed to recover in SOC medium containing caibenicillin. After recovery, the cells were held at 4°C until retrieved, after which an aliquot of cells were plated on LB agar supplemented with chloramphenicol, and kanamycin.
- replica patch plates were generated on two media types: 1) MacConkey agar base supplemented with lactose (as the sugar substrate), chloramphenicol, and kanamycin, and 2) MacConkey agar base supplemented with galactose (as the sugar substrate), chloramphenicol, and kanamycin. All liquid transfers were performed by the automated liquid handling device of the automated multi-module cell processing system.
- Cells are transfected with an editing cassette plasmid that mediates expression of a gene-specific gRNA with or without a DNA sequence to mediate precise genomic edits (HDR donor).
- This plasmid also expresses a handle to enable enrichment (cell surface receptor, fluorescent protein, antibiotic resistance gene) of cells that have been functionally transfected with the editing cassette plasmid.
- Cells are also co- transfected with nuclease (plasmid, mRNA, protein) that, when paired with the gene-specific gRNA can mediate DNA sequence specific endonuclease activity at genomic targets [00273]
- nuclease plasmid, mRNA, protein
- the enrichment handle After delivery of an enrichment-competent editing cassette, the enrichment handle must be expressed to levels that support specific positive selection of transfected cells while allowing for depletion of cells that did not receive an enrichment-competent editing cassette.
- the expression level of the enrichment reporter may enable enrichment of sub-populations that have significantly higher or lower levels of the enrichment reporter.
- Surface reporter-expressing cells can be specifically labeled using fluorophore- conjugated antibodies and then sorted into different populations (receptor-negative, high, or low) using a Fluorescence Activated Cell Sorter (FACS).
- FACS Fluorescence Activated Cell Sorter
- GFP-to-BFP analysis performed on the enriched populations versus unenriched populations
- certain subpopulations of enrichment of cells have demonstrated higher rates of editing as measured by the relative percentages, of GFP- positive, BFP-positive, and double-negative cells.
- Enrichment via cell-surface displayed receptors or affinity ligands has also been performed using antibody-coupled magnetic beads.
- An editing detection assay was developed using RNA-directed nuclease-GFP expression cassettes which expedites genome editing workflows from initial nuclease screening to the final stages of single cell cloning.
- This vector also included a U6- gRNA cassette creating a single vector system for CRISPR/nuclease delivery and expression (FIG. 10).
- the first system used a single-vector, with the co-expression of the RNA-directed nuclease ⁇ e.g., the Cas9 nuclease or the MAD7 nuclease) and GFP from the same mRNA, and a two-plasmid system in which the RNA- directed nuclease was expressed on a separate vector.
- the single vector system described here contained a T7 promoter for in vitro transcription of nuclease-GFP mRNA (FIG. 10).
- the ability to detect and enrich via GFP expression significantly reduces labor and cost associated with single cell cloning and genotyping in genome editing applications.
- the following data set illustrates how our single vector system can be used for expression monitoring and FACS enrichment of low and high level cutting.
- the single plasmid GFP format ensured that all required CRISPR/nuclease components (e.g. MAD7 and gRNA coding sequences) are effectively delivered to GFP positive cells.
- the GFP reporter allowed for quick detection of transfection efficiency saving time and cost associated with downstream expression quantification assays. This assay also allowed for rapid troubleshooting of plasmid delivery and expression problems associated with particular cell types. If GFP expression and nuclease indel activity cannot be observed in a particular cell type despite repeated attempts, using the nuclease-GFP mRNA can circumvent promoter / cell-type incompatibilities.
- a GFP to BFP reporter cell line was created using mammalian cells with a stably integrated genomic copy of the GFP gene (HEK293T-GFP). These cell lines enabled phenotypic detection of genomic edits of different classes (NHEJ, HDR, no edit) by various different mechanisms, including flow cytometry, fluorescent cell imaging, and genotypic detection by sequencing of the genome-integrated GFP gene. Lack of editing, or perfect repair of cut events in the GFP gene, result in cells that remain GFP-positive.
- Cut events that are repaired by the Non-Homologous End-Joining (NHEJ) pathway often result in nucleotide insertion or deletion events (indels), resulting in frame-shift mutations in the coding sequence that cause loss of GFP gene expression and fluorescence.
- Cut events that are repaired by the Homology-Directed Repair (HDR) pathway using the GFP to BFP HDR donor as a repair template, result in conversion of the cell fluorescence profile from that of GFP to that of BFP.
- HDR Homology-Directed Repair
- Thy 1.2 is a cell surface protein that is expressed on mouse thymocytes and not found on any human cells. Thy 1.2 is thus a unique reporter for identifying human cells that have received the editing machinery necessary to provide Thy 1.2 expression.
- 2x10 s cells were nucleofected with 200 ng of the MAD7 expression plasmid and 200 ng of the Thy 1.2-expressing GFP-to-BFP editing cassette using program CM- 130 on a 4D-Nucleofector X-unit (Lonza, Morristown, NJ) in 20 pL nucleocuvettes.
- Cells that were enriched for editing cassette uptake and Thyl.2 expression by FACS were 15-68% GFP-positive (WT), 30-74% GFP and BFP- negative (NHEJ), and 2-10% BFP-positive (HDR), depending on whether the low- expressing or high-expressing population was specifically enriched.
- Example V The enrichment methods as described above in Example V showed very similar efficiencies using magnetic-activated cell sorting (MACS) analysis.
- MCS magnetic-activated cell sorting
- 2x10 s cells were nucleofected with 200 ng of the MAD7 expression plasmid and 200 ng of the Thy 1.2-expressing GFP-to-BFP editing cassette using program CM- 130 on a 4D-Nucleofector X-unit (Lonza, Morristown, NJ) in 20 pL nucleocuvettes.
- Example VII ATetherin-HA -mediated enrichment for editing cassette untake mine FACS
- Cells with a stably integrated copy of the GFP gene were co-nucleofected with a plasmid expressing MAD7 nuclease and a GFP-to-BFP editing cassette plasmid that also drives expression of the cell surface ligand Tetherin that has been engineered to contain an additional His-tag and a deletion rendering the protein non-functional.
- the ATetherin-HA used is a cell-surface surrogate handle that contains a deletion rendering the molecule non-functional.
- 2x10 s cells were nucleofected with 200 ng of the MAD7 expression plasmid and 200 ng of the ATetherin-HA-expressing GFP-to-BFP editing cassette using program CM- 130 on a 4D-Nucleofector X-unit (Lonza, Morristown, NJ) in 20 pL nucleocuvettes.
- Example VIII Titration of receptor-specific magnetic beads to enrich for subpopulations of cells with higher reporter expression and editing rates
- Cells with a stably integrated copy of the GFP gene were co-nucleofected with a plasmid expressing MAD7 nuclease and a GFP-to-BFP editing cassette plasmid that also drives expression of the cell surface ligand ATetherin-HA or Thy 1.2
- 2x10 s cells were nucleofected with 200 ng of the MAD7 expression plasmid and 200 ng of the ATetherin-HA or Thy 1.2 -expressing GFP-to-BFP editing cassette using program CM- 130 for HEK293T or DS-120 for HAP1-GFP on a 4D-Nucleofector X-unit (Lonza, Morristown, NJ) in 20 pL nucleocuvettes.
- HEK293T-GFP cells enriched for editing machinery uptake using different amounts of Thy 1.2-specific MACS beads were re-plated into 24 well tissue culture plates and allowed to undergo gene editing and GFP to BFP conversion. As the amount of beads was increased, the proportion of cells with imprecise edits (GFP- and BFP- negative) and precise edits (BFP-positive) increased accordingly (FIG. 21).
- FACS FACS to specifically enrich HAP1 cells expressing high levels of ATetherin-HA.
- cells with a stably integrated copy of the GFP gene were co-nucleofected with one plasmid expressing MAD7 nuclease and an editing cassette that mediates a six base pair insertion into the DNMT3b gene and a second plasmid with a GFP-to-BFP editing cassette that also drives expression of the cell surface ligand Thy 1.2.
- 2x10 5 cells were nucleofected with 200 ng of the MAD7 expression plasmid and 200 ng of the Thy 1.2-expressing GFP-to-BFP editing cassette using program CM- 130 on a 4D-Nucleofector X-unit (Lonza, Morristown, NJ) in 20 pL nucleocuvettes.
- genomic DNA was purified from each subpopulation of enriched or unenriched cells using a Qiagen DNeasy blood and tissue kit (Velmo, Netherlands).
- a 613 base pair fragment of the DNMT3b gene was amplified by PCR with primers outside the region spanned by the 180 base pair homology arm regions on the editing cassette plasmid.
- a second PCR reaction was performed to amplify a 180 base pair region of DNMT3b gene containing the region targeted by the MAD7-gRNA complex and the 6 base insertion targeted by the HDR donor on the editing cassette.
- These PCR amplicons were prepared for NGS using an Dlumina TruSeq DNA sample prep kit according to the manufacturer’s directions.
- NGS analysis was performed using a custom NGS analysis and sequencing read alignment pipeline to bin read counts according to sequence identity to DNMT3b (WT) DNMT3b with a complete or partial targeted 6 base insertion (HDR_complete or HDR_partial) or a DNMT3b sequence containing insertions or deletions (Indel or NHEJ).
- WT DNMT3b
- HDR_complete or HDR_partial a complete or partial targeted 6 base insertion
- Indel or NHEJ a DNMT3b sequence containing insertions or deletions
- CeUs enriched for cassette uptake by MACS had insertions or deletions (Indel).
- Cells that were enriched for editing cassette uptake by MACS had 11.2% complete intended HDR-mediated knock-in edits, 1.3% partial HDR edits, and 78.4% Indels.
- ceUs that did not undergo any enrichment exhibited 4.2% complete intended HDR-mediated knock-in edits, 0.5% partial HDR edits, and 51.8% Indels. (FIG. 24).
- CREATE Fusion Editing is a novel technique that uses a nucleic acid nickase fusion protein having reverse transcriptase activity with a nucleic acid encoding a gRNA comprising a region complementary to a target region of a nucleic acid in one or more cells covalently linked to an editing cassette comprising a region homologous to the target region in the one or more cells with a mutation of at least one nucleotide relative to the target region in the one or more cells and a protospacer adjacent motif (PAM) mutation.
- PAM protospacer adjacent motif
- a nickase enzyme derived from a Type II CRISPR enzyme was fused to an engineered reverse transcriptase (RT) on the C-terminus and cloned downstream of a CMV promoter.
- RT reverse transcriptase
- M-MLV Moloney Murine Leukemia Virus
- CFE2.1 CREATE Fusion Editor 2.1
- CFE2.2 an enrichment handle (T2A-dsRed) was also added on the C-terminus of CFE2.1. The enrichment handle allowed selection of the cells that express the nickase enzyme and RT fusion protein.
- RNA guides were designed that were complementary to a single region proximal to the EGFP-to-BFP editing site.
- the CREATE Fusion gRNA was extended on the 3’ end to include a region of 13 bp that include the TY-to-SH edit and a second region of 13 bp that is complementary to the nicked EGFP DNA sequence (FIG. 26). This allows the nicked genomic DNA to anneal to the 3’ end of the gRNA which can then be extended by the RT to incorporate the edit in the genome.
- the second gRNA targets a region in the EGFP DNA sequence that is 86 bp upstream of the edit site.
- This gRNA was designed such that it enables the nickase to cut the opposite strand relative to CREATE Fusion gRNA. Both of these gRNAs were cloned downstream of a U6 promoter. A poly T sequence was also included that terminates the transcription of the gRNA.
- FIG. 27 A flow chart of the exemplary experimental process carried out is shown in FIG. 27.
- the plasmids were transformed into NEB Stable E. coli (Ipswich, NY) and grown overnight in 25 mL LB cultures. The following day the plasmids were purified from E. coli using the Qiagen Midi Prep kit (Venlo, Netherlands). The purified plasmid was then RNase A (ThermoFisher, Waltham, Mass) treated and re-purified using the DNA Clean and Concentrator kit (Zymo, Irvine, CA).
- HEK293T cells were cultured in DMEM medium which was supplemented with 10% FBS and IX Penicillin and Streptomycin. 100 ng of total DNA (50 ng of gRNA plasmid and 50 ng of CFE plasmids) was mixed with 1 ⁇ l of PolyFect (Qiagen, Venlo, Netherlands) in 25 ⁇ l of OptiMEM in a 96 well plate. The complex was incubated for 10 minutes and then 20,000 HEK293T cells resuspended in 100 ⁇ l of DMEM were added to the mixture. The resulting mixture was then incubated for 80 hours at 37 C and 5% CO 2 .
- the cells were harvested from flat bottom 96 well plates using TrypLE Express reagent (ThermoFisher, Waltham, Mass) and transferred to v-bottom 96 well plate. The plate was then spun down at 500 g for 5 minutes. The TrypLE solution was then aspirated and the cell pellet was resuspended in FACS buffer (IX PBS, 1 % FBS, 1 mM EDTA and 0.5% BSA). The GFP+, BFP+ and RFP+ cells were then analyzed on the Attune NxT flow cytometer and the data was analyzed on FlowJo software.
- the RFP+BFP+ cells that were identified were indicative of the proportion of enriched cells that have undergone precise or imprecise editing process.
- BFP+ cells indicate cells that have undergone successful editing process and express BFP.
- the GFP- cells indicate cells that have been imprecisely edited, leading to disruption of the GFP open reading frame and loss of expression.
- the CREATE Fusion Editing process utilized a gRNA covalently linked to a region of homology to the intended target site in the genome.
- the edit is immediately 3 ’ of the gRNA, and 3 ’ of the edit is a further region complementary to the nicked genome, although the intended edit could also be present further 5’ within the region homologous to the nicked genome.
- a nickase RT fusion enzyme created a nick in the target site and the nicked DNA annealed to its complementary sequence on the 3’ end of the gRNA. The RT then extended the DNA, thereby incorporating the intended edit directly in the genome.
- An enrichment handle specifically a fluorescent reporter (RFP) linked to nuclease expression, (CFE2.2) was included in this experimentation as a proxy for cells receiving the editing machinery.
- RFP fluorescent reporter
- CFE2.2 nuclease expression
- Cells with a stably integrated copy of the GFP gene were nucleofected with a plasmid expressing MAD7 nuclease and a GFP-to-BFP editing cassette plasmid that also drives expression of a fluorescent reporter molecule (dsRed) or a CREATE-Fusion enzyme plasmid with an RFP reporter (FIG. 25, CPE2.2) and a CREATE-Fusion gRNA expressing plasmid driving nick-based editing of GFP to BFP (FIG. 26, GFP CREATE’).
- dsRed fluorescent reporter molecule
- CREATE-Fusion enzyme plasmid with an RFP reporter FIG. 25, CPE2.2
- CREATE-Fusion gRNA expressing plasmid driving nick-based editing of GFP to BFP FIG. 26, GFP CREATE’
- lxlO 6 cells were nucleofected with 4 ug of the MAD7 GFP to BFP editing plasmid or 2 ug the CREATE-Fusion enzyme plasmid and 2 ug of the CREATE-Fusion gRNA plasmid using program CM- 130 on a 4D- Nucleofector X-unit (Lonza, Morristown, NJ) in 100 ⁇ L nucleocuvettes.
- the FACS-sorted subpopulations, as well as an unenriched control sample were plated in separate wells of a 24-well tissue culture dish and allowed to undergo gene-editing.
- the cells receiving a knock-in edit display a GFP-to-BFP conversion phenotype.
- CREATE Fusion Editing was carried out in mammalian cells using a single guide RNA covalently linked to a homology arm having an intended edit to the native sequence and an edit that disrupts nuclease cleavage at this site.
- the basic protocol is set forth in FIG. 32.
- lentiviral vectors were produced using the following protocol:_1000 ng of Lentiviral transfer plasmid containing the CREATE Fusion cassettes (FIGs. 23 and 24) along with 1500 ng of Lentiviral Packaging plasmids (ViraSafe Lentivirus Packaging System Cell BioLabs) were transfected into HEK293T cells using Lipofectamine LTX in 6-well plates. Media containing the lentivirus was collected 72 hrs post transfection. Two clones of a lentiviral CREATE Fusion gRNA-HA design were chosen, and an empty lentiviral backbone was included as negative control.
- FIGs 35A and 35B A simple example of how this can be implemented is shown in FIGs 35A and 35B.
- a CREATE fusion enzyme comprising the nickase and RT activities is encoded on the same plasmid or amplicon as a dual CREATE cassette fusion system (FIG.35 A).
- CREATE cassette 1 encodes the gRNA-HA targeting sequences that once transcribed into RNA are necessary to guide nick-translation based editing at a functional site of interest in the chromosome.
- CREATE cassette 2 encodes a second gRNA-HA set that targets an inert secondary site, for example the 3’ UTR of a pseudogene as one possible location to integrate a DNA barcode that is unique for each target site variant.
- the covalent coupling of the gRNA-HA elements within each editing cassette function to colocalize the RNA for efficient reverse transcription at each nick site to drive the editing process at each locus. Meanwhile the covalent coupling between cassettes ensures the two edits are highly correlated at the single cell level.
- the unique identity of the barcode sequence encoded in CREATE cassette 2 once integrated, thus serves as a trackable genomic barcode that can report the identity of edits across the genome based on sequencing or other molecular readouts of a fixed chromosomal position. This barcoding approach reduces the complexity of downstream population sequencing to simple PCR amplicon sequencing assays.
- this recording logic can be further expanded to cover combinatorial edits within a single cell by the inclusion of additional CREATE cassettes (FIG 35B).
- the recording site and unique barcode are maintained, but the editing sites encompass >2 targets within the same cell.
- the barcode now provides a report of combinatorial editing events on a single cell level and allows fitness tracking and computational de-convolution of combinatorial edited cell populations using the trackable barcode feature.
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