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WO2018039783A1 - Systèmes et procédés de sélection pour l'édition génomique - Google Patents

Systèmes et procédés de sélection pour l'édition génomique Download PDF

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WO2018039783A1
WO2018039783A1 PCT/CA2017/051011 CA2017051011W WO2018039783A1 WO 2018039783 A1 WO2018039783 A1 WO 2018039783A1 CA 2017051011 W CA2017051011 W CA 2017051011W WO 2018039783 A1 WO2018039783 A1 WO 2018039783A1
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amino acid
cells
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sequence
ouabain
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Yannick Doyon
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Universite Laval
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to genome editing. More specifically, the present invention is concerned with reagents, products and methods for modifying the genome of a cell and for selecting and isolating cells bearing the targeted modification.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • ZFNs zinc-finger nucleases
  • TALENs TAL effector nucleases
  • CRISPR-based technologies enable researchers to introduce double-strand breaks (DSBs) into virtually any DNA sequence using sequence-specific single guide RNAs (sgRNAs) that target Cas9 and Cpf1 nucleases to cleave the matching target.
  • sgRNAs sequence-specific single guide RNAs
  • multiple DSBs can be created in the same cell via expression of distinct sgRNAs, a process referred to as multiplexing (5).
  • the ensuing DSBs activate two competing repair pathways, namely non-homologous end joining (NHEJ) and homology-directed repair (HDR) to facilitate the precise modification of the targeted endogenous locus.
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Fluorescence-based surrogate target gene reporters have also been successfully used to enrich for cells with high nuclease activity (14, 15).
  • a limitation of both methods is that single-cell FACS enrichment is not possible for certain sensitive cell lines whereas construction of a surrogate reporter for each target site decreases the throughput of genome editing.
  • enhancing HDR-mediated processes by altering cell cycle parameters, the timing of nuclease expression, chemical inhibition of NHEJ and use of HDR agonists show promising results but their general applicability and effects on specificity need to be thoroughly evaluated (16-22).
  • the present invention makes use of an endogenous gene and a drug (e.g., a highly potent small molecule (e.g., cardiotonic steroids such as ouabain)) to prepare and rapidly select and enrich for genome edited cells.
  • a drug e.g., a highly potent small molecule (e.g., cardiotonic steroids such as ouabain)
  • the process does not involve the introduction of superfluous "exogenous" DNA material.
  • the method disclosed herein has the advantage of using a selection agent (ouabain or ouabain derivatives) that is non-mutagenic.
  • the strategy employed was shown to provide the unexpected advantage of permitting the selection of NHEJ or HDR-based gene modification (i.e., favoring NHEJ or HDR-based gene modification), the latter being a highly sought-for characteristic in the field.
  • the genome editing approach described herein is based in part on Na+,K+-ATPase and inhibitors thereof, notably cardiotonic steroids such as ouabain.
  • Cardiotonic steroids such as ouabain (PubChem CID: 439501), constitute a broad class of specific Na+,K+-ATPase inhibitors prescribed for congestive heart failure for more than 2 centuries (29).
  • Ouabain is a highly potent plant-derived inhibitor of the Na+,K+-ATPase (Enzyme Entry E.C. 3.6.1.96) (29-31).
  • the Na+,K+-ATPase encoded by the ATP1A1 gene (GeneCards ID: GC01 P116372; HGNC: 799; Entrez Gene: 476; Ensembl: ENSG00000163399; OMIM: 182310; UniProtKB: P05023) is the ion pump responsible for maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane of animal cells. It is an essential and ubiquitously expressed enzyme functioning as a heteromeric complex consisting of a large a-subunit (ATP1 A1), which is responsible for ATP hydrolysis and ion transport, and a ⁇ -subunit, acting as a chaperone (29, 30).
  • ATP1 A1 a large a-subunit
  • ⁇ -subunit acting as a chaperone
  • the a1 isoform is ubiquitously expressed, whereas the a2 isoform is expressed mainly in skeletal, heart, and smooth muscle, brain, lung, and adipocytes.
  • the a3 isoform occurs mainly in neurons and ovaries as well as in developing hearts of rat and in adult human heart. This isoform also occurs in white blood cells.
  • the a4 isoform of the Na,K-ATPase is found in sperm. In humans, all ATP1A isoforms are sensitive to ouabain and specific mutations within the ATP1 A1 isoform have been described to confer resistance to the drug. Based on the sequence conservation of ATP1A isoforms (FIG.
  • gain-of-function alleles in an endogenous gene e.g., ATP1A1
  • an endogenous gene e.g., ATP1A1
  • selected drugs e.g., ouabain
  • the present invention is useful for increasing the efficiency of selection, enrichment and identification of cells in which genome editing has likely occurred. It can be used in basic research contexts as well as for therapeutic applications.
  • the present invention relates to nucleases and methods derived therefrom for the targeted modification of an endogenous gene locus (e.g., an ATP1 A gene locus, e.g., ATP1A1 , ATP1A2, ATP1A3 or ATP1A4) and for increasing the frequency of gene modification at a second, target polynucleotide locus.
  • an endogenous gene locus e.g., an ATP1 A gene locus, e.g., ATP1A1 , ATP1A2, ATP1A3 or ATP1A4
  • a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells and for enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population comprising:
  • HDR homology-directed repair
  • NHEJ non-homologous end joining
  • the mutations are introduced by nuclease(s) specific for the target polynucleotide and endogenous gene locus (e.g., CRISPR nucleases, TALENs, Meganucleases, zinc finger nucleases, etc.).
  • nuclease(s) specific for the target polynucleotide and endogenous gene locus e.g., CRISPR nucleases, TALENs, Meganucleases, zinc finger nucleases, etc.
  • the nuclease-based modification in the target polynucleotide (the first mutation) in methods of the present invention is introduced by HDR and step (i) further comprises introducing into the one or more target cells a donor nucleic acid comprising the modification.
  • the nuclease-based modification in the endogenous gene locus (the second mutation) in methods of the present invention is introduced by HDR and step (i) further comprises introducing into the one or more target cells a second donor nucleic acid comprising the second modification.
  • introducing a mutation in the endogenous gene locus involves introducing one or more double-stranded breaks (DSBs) in an exon of the gene locus using a nuclease specific for the gene locus (e.g., a zinc finger nuclease, a Meganuclease, a TALEN or a CRISPR nuclease).
  • a nuclease specific for the gene locus e.g., a zinc finger nuclease, a Meganuclease, a TALEN or a CRISPR nuclease.
  • introducing a mutation in the endogenous gene locus involves introducing a double-stranded break (DSB) in an intron of the gene locus using a nuclease specific for the gene locus (e.g., a zinc finger nuclease, a meganuclease, a TALEN or a CRISPR nuclease).
  • a nuclease specific for the gene locus e.g., a zinc finger nuclease, a meganuclease, a TALEN or a CRISPR nuclease.
  • the present invention further provides a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells and for enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population, the method comprising:
  • a sgRNA guide sequence having a target sequence in an endogenous gene such that the sgRNA guide sequence directs the cleavage of the endogenous gene for introduction of a mutation (i.e., a second nuclease-based modification) in the endogenous gene conferring resistance to a drug, and a CRISPR nuclease recognition sequence;
  • sgRNA guide sequence having a target sequence in the target polynucleotide and a CRISPR nuclease recognition sequence
  • target sequences in (a) and (b) are contiguous to a protospacer adjacent motif (PAM) recognized by the CRISPR nuclease;
  • PAM protospacer adjacent motif
  • the present invention further provides a method of inducing drug resistance in one or more target cells within a population of cells, the method comprising:
  • the present invention further provides a method for enriching for target cells modified at an endogenous gene locus, wherein the modification confers resistance to a drug, the method comprising:
  • the endogenous gene and drug are selected from the endogenous genes and corresponding drugs set forth in Table 1 herein.
  • the endogenous gene and associated drug is selected from the genes and drugs listed in Table 1.
  • the endogenous gene is (encodes) a Na/K-ATPAse and the drug is an Na/K- ATPase inhibitor.
  • the endogenous gene is (encodes) an ATP1A gene, and the drug is ouabain or a derivative thereof.
  • the endogenous gene is (encodes) MGMT and the drug is 06- benzylguanine.
  • the ouabain or a derivative thereof is used for selection (i.e., is comprises in the selection medium) at a concentration of less than about 5 ⁇ , in a further embodiment at a concentration of about 100 ⁇ or less, in a further embodiment at a concentration of about 1000 ⁇ (1 mM) or less, in a further embodiment from about 5 ⁇ to about 100 ⁇ , in a further embodiment from about 5 ⁇ to about 1000 ⁇ , in a further embodiment from about ⁇ ⁇ about 1000 ⁇ , in a further embodiment from about 5 ⁇ to about 500 ⁇ , in a further embodiment from about 100 ⁇ to about 500 ⁇ , in a further embodiment from about 5 ⁇ to about 800 ⁇ , in a further embodiment from about 100 ⁇ to about 800 ⁇ in a further embodiment from about 75 ⁇ to about 125 ⁇ , in a further embodiment from about 75 ⁇ to about 500 ⁇ , in a further embodiment from about 75 ⁇ to about 800 ⁇ , in a further embodiment from about 75 ⁇ to about 1000 ⁇ .
  • the present invention further provides a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells comprising: (i) introducing (e.g., (co- introducing), by HDR or NHEJ, a mutation (a nuclease-based modification) in (a) an ATP1A gene locus and (b) the target polynucleotide, in the one or more target cells; (ii) contacting the population of cells with ouabain or a derivative thereof; and (iii) selecting the one or more target cells by virtue of increased tolerance to ouabain or derivative thereof, thereby enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population of cells.
  • introducing e.g., (co- introducing), by HDR or NHEJ, a mutation (a nuclease-based modification) in (a) an ATP1A gene locus and (b) the target
  • the present invention further provides a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells comprising: (i) providing the cell population with a CRISPR nuclease and one or more sgRNAs comprising: (a) a sgRNA guide sequence having a target sequence in the ATP1A gene and a CRISPR nuclease recognition sequence; (b) a sgRNA guide sequence having a target sequence in the target polynucleotide and a CRISPR nuclease recognition sequence; wherein the target sequences in (a) and (b) are contiguous to a protospacer adjacent motif recognized by the CRISPR nuclease; (ii) contacting the population of cells with ouabain or a derivative thereof; and (iii) selecting the one or more target cells by virtue of increased tolerance to ouabain or derivative thereof, thereby enriching the number of target cells comprising the mutation (CRISPR
  • the present invention further provides a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells and for enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population comprising: (i) introducing, by HDR or NHEJ, a mutation (nuclease-based modification) in (a) an ATP1A gene locus and (b) the target polynucleotide, in the one or more target cells; (ii) contacting the population of cells with ouabain or a derivative thereof; and (iii) selecting the one or more target cells by virtue of increased tolerance to ouabain or derivative thereof, thereby enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population of cells.
  • the present invention further provides a method for nuclease-based modification of a target polynucleotide in one or more target cells within a population of cells and for enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide within the population comprising: (i) providing the cell population with a CRISPR nuclease and one or more sgRNAs comprising: (a) a sgRNA guide sequence having a target sequence in the ATP1A gene and a CRISPR nuclease recognition sequence; (b) a sgRNA guide sequence having a target sequence in the target polynucleotide and a CRISPR nuclease recognition sequence; wherein the target sequences in (a) and (b) are contiguous to a protospacer adjacent motif recognized by the CRISPR nuclease; (ii) contacting the population of cells with ouabain or a derivative thereof; and (iii) selecting the one or
  • the present invention further provides a method of inducing ouabain resistance in one or more target cells within a population of cells, the method comprising: (i) introducing a double stranded break with a nuclease in the one or more target cells; (ii) contacting the cell population with ouabain or a derivative thereof; and (iii) selecting the one or more target cells by virtue of increased tolerance to ouabain or derivative thereof, thereby inducing ouabain resistance in the one or more target cells.
  • the present invention further provides a method for enriching for target cells modified at an ATP1 A gene locus, the method comprising: (i) cleaving with a nuclease the ATP1A gene in one or more target cells within a population of cells; (ii) contacting the population of cells with ouabain or a derivative thereof; and (iii) selecting the one or more target cells by virtue of increased tolerance to ouabain or derivative thereof, thereby enriching for target cells in which the ATP1 A gene has been modified.
  • the nuclease is a CRISPR nuclease
  • the method comprises providing the one or more target cells with a sgRNA comprising a sgRNA guide sequence having a target sequence in the ATP1 A gene and a CRISPR nuclease recognition sequence.
  • the CRISPR nuclease is Cas9 or Cpf1 nuclease.
  • the Cas9 is SpCas9 (or a functional derivative thereof) from Streptococcus pyogenes.
  • the Cpf1 is AsCpfl from Acidaminococcus sp.
  • the ATP1 A gene is ATP1 A2 and the sgRNA target sequence is selected within SEQ ID NO: 459.
  • the ATP1A gene is ATP1A3 and the sgRNA target sequence is selected within SEQ ID NO: 461.
  • the ATP1A gene is ATP1A4 and the sgRNA target sequence is selected within SEQ ID NO: 463.
  • the sgRNA target sequence is selected within a region of the ATP1A2, ATP1A 3 or ATP1A4 gene corresponding to exon 4 of ATP1A (see FIG. 37).
  • the ATP1A gene is ATP1 A1 and the sgRNA target sequence comprises or consists of a sequence as set forth in Table 3 and FIG. 28.
  • the sgRNA comprises or consists of the sequence set forth in any one of SEQ ID NOs: 24-26 (sgRNA G2, G3, G4) and 33-36 (sgRNA G6, G7, G8, G9).
  • the sgRNA target sequence is within an exon of the ATP1 A gene. In embodiments, the gRNA target sequence is within an intron of the ATP1 A gene.
  • the ATP1A gene is ATP1A1 and the exon is exon 4.
  • the target sequence comprises or consists of the following target sequence in the ATP1A1 gene: (i) ATCCAAGCTGCTACAGAAG (G2); (ii) GTTCCTCTTCTGTAGCAGCT (G4); (iii) TTGGCTTATAGCATCCAAGC (G6); (iv) AGGTTCCTCTTCTGTAGCAG (G7); or (v) GAGGTTCCTCTTCTGTAGCA (G8).
  • the ATP1A gene is ATP1A1 and the intron is intron 3 or intron 4.
  • the target sequence comprises or consists of the following target sequence on the ATP1A1 gene: (i) GAGTTCTGTAATTCAGCATA (G3) or (ii) TAGTACACATCAGATATCTT (G9).
  • methods of the present invention further comprise providing the cell with an ATP1A donor nucleic acid conferring ouabain resistance.
  • the ATP1A donor nucleic acid conferring ouabain resistance is an ATP1A1 donor nucleic acid.
  • the ATP1A1 donor nucleic acid comprises or consists of the following nucleic acid sequence: (i) CAATGTTACTGTGGATTGGAGCGATTCTTTGTTTCTTGGCTTATAGCATCGATGCTGCTACAGAAGAGGAACCT CAAAACGATCGTGTGAGTTCTGTAATTCAGCATATCGATTTGTAGTACACATCAGATATCTT (DR); or (ii) CAATGTTACTGTGGATTGGAGCGATTCTTTGTTTCTTGGCTTATAGCATCAGAGCTGCTACAGAAGAGGAACCT CAAAACGATGACGTGAGTTCTGTAATTCAGCATATCGATTTGTAGTACACATCAGATATCTT (RD).
  • the ATP1A1 donor nucleic acid confers ouabain resistance by replacing at least one of amino acids Q118 and N129 in the ATP1A1 gene by a charged amino acid. In embodiments, the ATP1A1 donor nucleic acid confers ouabain resistance by replacing amino acid Q118 by an aspartic acid and amino acid N129 by an arginine. In embodiments, the ATP1A1 donor nucleic acid confers ouabain resistance by replacing amino acid Q118 by an arginine and amino acid N129 by an aspartic acid.
  • the above-mentioned one or more target cells are selected from the group consisting of T-cells, B-cells or stem cells.
  • the stem cells comprise embryonic stem cells (ESCs), induced pluripotent stem cell (iPSCs), CD34+ hematopoietic stem cells or hepatic stem cells.
  • the one or more target cells are primary cells.
  • the present invention provides a ouabain resistant ATP1A polypeptide.
  • the ouabain resistant ATP1A polypeptide is a ouabain resistant ATP1A1 polypeptide.
  • the ouabain resistant ATP1A polypeptide is a ouabain resistant ATP1A2 polypeptide.
  • the ouabain resistant ATP1A polypeptide is a ouabain resistant ATP1A3 polypeptide.
  • the ouabain resistant ATP 1 A polypeptide is a ouabain resistant ATP 1A4 polypeptide.
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of an amino acid sequence as set forth in SEQ ID NOs: 5, 8, 10, 12, 14, 16, 18, 20 or 22.
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 5, wherein: (i) the amino acid at position 119 is any amino acid except an alanine or is absent; (ii) the amino acid at position 120 is any amino acid except an alanine or is absent; (iii) the amino acid at position 121 is any amino acid except a threonine or is absent; (iv) the amino acid at position 122 is any amino acid except a glutamic acid or is absent; (v) the amino acid at position 123 is any amino acid except a glutamic acid or is absent; (vi) the amino acid at position 124 is any amino acid except a glutamic acid or is absent; (vii) the amino acid at position 125 is any
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 6, wherein: (i) the amino acid at position 119 is any amino acid except an alanine or is absent; (ii) the amino acid at position 120 is any amino acid except an alanine or is absent; (iii) the amino acid at position 121 is any amino acid except a threonine or is absent; (iv) the amino acid at position 122 is any amino acid except a glutamic acid or is absent; (v) the amino acid at position 123 is any amino acid except a glutamic acid or is absent; (vi) the amino acid at position 124 is any amino acid except a glutamic acid or is absent; (vii) the amino acid at position 125 is any amino acid except a proline or a lysine or is absent; (viii) the amino acid at position 126 is any amino acid except a glutamine or is absent; (ix) any combination of (i),
  • the ouabain resistant ATP1 A1 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 5 or 6, wherein: (i) the amino acid at position 119 is any amino acid except an alanine or an isoleucine, or is absent; (ii) the amino acid at position 120 is any amino acid except an alanine, a glycine or a tyrosine, or is absent; (iii) the amino acid at position 121 is any amino acid except a threonine, a methionine or a phenylalanine, or is absent; (iv) the amino acid at position 122 is any amino acid except a glutamic acid or an asparagine, or is absent; (v) the amino acid at position 123 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vi) the amino acid at position 124 is any amino acid except a glutamic acid or an aspartic acid
  • the ouabain resistant ATP1 A1 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 5 or 6, wherein (i) the amino acid at position 119 is absent; (ii) the amino acid at position 120 is absent; (iii) the amino acid at position 121 is a lysine or is absent; (iv) the amino acid at position 122 is absent; (v) the amino acid at position 123 is absent; (vi) the amino acid at position 124 is absent; (vii) the amino acid at position 125 is an alanine or is absent; (viii) the amino acid at position 126 is absent; or (ix) any combination of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • the present invention provides an ATP1A1 ouabain resistant polypeptide, wherein the polypeptide comprises at least one deletion or non-conservative substitution at amino acid: (i) A119, A120, T121 , E122, E123, E124, P125 and/or Q126 of SEQ ID NO: 3 or 4.
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 6, wherein the polypeptide comprises at least one deletion or non-conservative substitution at amino acid: (i) A119, A120, T121 , E122, E123, E124, P125 and/or Q126.
  • the ouabain resistant ATP1A1 polypeptide further comprises at least one further amino acid mutation set forth in Table 4.
  • the present invention provides an ATP1A2 ouabain resistant polypeptide, wherein the polypeptide comprises at least one deletion or non-conservative substitution at amino acid A117, A118, M119, E120, D121 , E122, P123 and/or S124 of SEQ ID NO: 460.
  • the ouabain resistant ATP1A2 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 485, wherein: (i) the amino acid at position 117 is any amino acid except an alanine or an isoleucine, or is absent; (ii) the amino acid at position 118 is any amino acid except an alanine, a glycine or a tyrosine, or is absent; (iii) the amino acid at position 119 is any amino acid except a threonine, a methionine or a phenylalanine, or is absent; (iv) the amino acid at position 120 is any amino acid except a glutamic acid or an asparagine, or is absent; (v) the amino acid at position 121 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vi) the amino acid at position 122 is any amino acid except a glutamic acid or an aspartic acid, or is absent;
  • the ouabain resistant ATP1 A2 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 485, wherein (i) the amino acid at position 117 is absent; (ii) the amino acid at position 118 is absent; (iii) the amino acid at position 119 is a lysine or is absent; (iv) the amino acid at position 120 is absent; (v) the amino acid at position 121 is absent; (vi) the amino acid at position 122 is absent; (vii) the amino acid at position 123 is an alanine or is absent; (viii) the amino acid at position 124 is absent; or (ix) any combination of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • the present invention provides an ATP1A3 ouabain resistant polypeptide, wherein the polypeptide comprises at least one deletion or non-conservative substitution at amino acid: A109, G110, T111 , E112, D113, D114, P115 and/or S116 of SEQ ID NO: 462.
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of a polypeptide as set forth in 486, wherein: (i) the amino acid at position 109 is any amino acid except an alanine or an isoleucine, or is absent; (ii) the amino acid at position 110 is any amino acid except an alanine, a glycine or a tyrosine, or is absent; (iii) the amino acid at position 111 is any amino acid except a threonine, a methionine or a phenylalanine, or is absent; (iv) the amino acid at position 112 is any amino acid except a glutamic acid or an asparagine, or is absent; (v) the amino acid at position 113 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vi) the amino acid at position 114 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vii) the amino acid at position
  • the ouabain resistant ATP1 A3 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 486, wherein (i) the amino acid at position 109 is absent; (ii) the amino acid at position 110 is absent; (iii) the amino acid at position 111 is a lysine or is absent; (iv) the amino acid at position 112 is absent; (v) the amino acid at position 113 is absent; (vi) the amino acid at position 114 is absent; (vii) the amino acid at position 115 is an alanine or is absent; (viii) the amino acid at position 116 is absent; or (ix) any combination of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • the present invention provides an ATP1A4 ouabain resistant polypeptide, wherein the polypeptide comprises at least one deletion or non-conservative substitution at amino acid: 1127, Y128, F129, N130, E131 , E132, P133 and/or T134 of SEQ ID NO: 464.
  • the ouabain resistant ATP1A1 polypeptide comprises or consists of a polypeptide as set forth in 486, wherein: (i) the amino acid at position 127 is any amino acid except an alanine or an isoleucine, or is absent; (ii) the amino acid at position 128 is any amino acid except an alanine, a glycine or a tyrosine, or is absent; (iii) the amino acid at position 129 is any amino acid except a threonine, a methionine or a phenylalanine, or is absent; (iv) the amino acid at position 130 is any amino acid except a glutamic acid or an asparagine, or is absent; (v) the amino acid at position 131 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vi) the amino acid at position 132 is any amino acid except a glutamic acid or an aspartic acid, or is absent; (vii) the amino acid at position
  • the ouabain resistant ATP1 A4 polypeptide comprises or consists of a polypeptide as set forth in SEQ ID NO: 487, wherein (i) the amino acid at position 127 is absent; (ii) the amino acid at position 128 is absent; (iii) the amino acid at position 129 is a lysine or is absent; (iv) the amino acid at position 130 is absent; (v) the amino acid at position 131 is absent; (vi) the amino acid at position 132 is absent; (vii) the amino acid at position 133 is an alanine or is absent; (viii) the amino acid at position 134 is absent; or (ix) any combination of (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • the present invention also provides a sgRNA for inducing or conferring ouabain resistance by NHEJ or HDR comprising (i) a sgRNA guide sequence having a target sequence in an ATP1A gene; and (ii) a CRISPR nuclease recognition sequence.
  • the sgRNA guide sequence has a target sequence in the ATP1A1 gene. In embodiments, the sgRNA guide sequence has a target sequence comprising or consisting of a target sequence (without the PAM) as set forth in Table 3 and FIG. 28.
  • the sgRNA comprises a sgRNA guide sequence having a target sequence comprising or consisting of the following target sequence in the ATP1 A1 gene: (i) GATCCAAGCTGCTACAGAAG (G2, SEQ ID NO: 28); (ii) GTTCCTCTTCTGTAGCAGCT (G4, SEQ ID NO: 30); (iii) TTGGCTTATAGCATCCAAGC (G6, SEQ ID NO: 38); (iv) AGGTTCCTCTTCTGTAGCAG (G7, SEQ ID NO: 39); or (v) GAGGTTCCTCTTCTGTAGCA (G8, SEQ ID NO: 40).
  • the sgRNA guide sequence has a target sequence comprising or consisting of the following target sequence in the ATP1A1 gene: (i) GAGTTCTGTAATTCAGCATATGG (G3, SEQ ID NO: 29) or (ii) TAGTACACATCAGATATCTT (G9, SEQ ID NO: 41).
  • the present invention also provides nucleic acids encoding drug resistant polypeptides (e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1A polypeptides) and sgRNAs described herein, vectors for expressing nucleic acids of the present invention and cells comprising such vectors and/or nucleic acids.
  • the vector is a viral vector.
  • the present invention also provides a kit comprising one or more: sgRNAs; drug resistant polypeptides (e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1A polypeptides, etc.); nucleic acids, vectors; cells and/or donor nucleic acids (e.g., donor nucleic acids) described herein.
  • the kit further comprises instructions to use the kit according to methods disclosed herein and/or drug(s) for selecting (enriching) cells expressing drug resistant polypeptides generated using methods disclosed herein.
  • the present invention also relates to the use of one or more sgRNAs; drug resistant polypeptides (e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1A polypeptides, etc.); nucleic acids, vectors; cells, donor nucleic acids and/or kits described herein for (a) inducing or conferring drug resistance (e.g., Na/K ATPase inhibitor resistance, ouabain resistance, etc.) in one or more target cells; (b) nuclease-based modification of a target polynucleotide in one or more target cells and for enriching the number of target cells comprising the nuclease- based modification of the target polynucleotide; and/or or (c) for enriching for target cells modified by a nuclease at a drug resistance gene locus (e.g., an Na/K ATPase gene locus, ATP1 A gene locus, MGMT gene locus, etc.).
  • the present invention also provides one or more sgRNAs; drug resistant polypeptides (e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1A polypeptides, etc.); nucleic acids, vectors; cells and/or donor nucleic acids described herein for use in (a) inducing or conferring drug resistance (e.g., Na/K ATPase inhibitor resistant, ouabain resistance etc.) in one or more target cells; (b) for nuclease-based modification of a target polynucleotide in one or more target cells for enriching the number of target cells comprising the nuclease-based modification of the target polynucleotide; and/or or (c) for enriching for target cells modified by a nuclease at a drug resistance gene locus (e.g., an ATP1A gene locus).
  • drug resistant polypeptides e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1
  • the present invention also provides a composition comprising one or more sgRNAs; drug resistant polypeptides (e.g., Na/K ATPase inhibitor resistant, ouabain resistant ATP1A polypeptides, 06-benzylguanine resistant, etc.); nucleic acids, vectors; cells and/or donor nucleic acids described herein.
  • the composition may further comprise a suitable carrier (e.g., pharmaceutically acceptable).
  • the present invention further concerns the above-described composition or kit for (a) inducing or conferring drug resistance (e.g., Na/K ATPase inhibitor resistance, ouabain resistance, 06-benzylguanine resistance, etc.) in one or more target cells; (b) for modifying with a nuclease a target polynucleotide in one or more target cells and for enriching the number of target cells comprising the modified target polynucleotide; and/or or (c) for enriching for target cells modified by a nuclease at an ATP1 A gene locus.
  • drug resistance e.g., Na/K ATPase inhibitor resistance, ouabain resistance, 06-benzylguanine resistance, etc.
  • the above-mentioned ATP1A gene is an ATP1A1 , ATP1A2, ATP 1 A3, or ATP1A4 gene.
  • the ATP1A gene is ATP1A1.
  • FIG. 1 shows a schematic representation of SpCas9 target sites surrounding DNA encoding the first extracellular loop of human ATP1A1 (SEQ ID NOs: 1 and 3). Annotated are the positions of residues Q118 and N129, exon/intron boundary, protospacer adjacent motifs (PAM) and four potential SpCas9 target sequences (Targets 1-4, SEQ ID NOs: 59 (Target 1), 70 (Target 2), 29 (Target 3), and 30 (Target 4));
  • FIG. 2 shows the identification of highly active sgRNA/nuclease combinations targeting the ATP1A1 locus.
  • the indicated single-guide RNA (sgRNA G1-G4: SEQ ID NOs: 23-26) expression vectors (500 ng) were transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 (adeno-associated virus integration site 1 ; 19q13-qter; HGNC:22; OMIM: 102699; GenBank: S51329.1) safe harbor locus.
  • AAVS1 adeno-associated virus integration site 1 ; 19q13-qter; HGNC:22; OMIM: 102699; GenBank: S51329.1
  • Genomic DNA was harvested 3 and 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control.
  • FIG. 3 shows active sgRNAs/nucleases targeting the coding sequence surrounding Q118 and N129 of ATP1A1 induce cellular resistance to ouabain.
  • the indicated single-guide RNA (sgRNA, G1-G4: SEQ ID NOs: 23- 26) expression vectors 500 ng were transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus. Cells were treated with 0.5 ⁇ ouabain for 7 days starting 3 days post transfection. Only cells transfected with sgRNA G2 (SEQ ID NO: 24) and G4 (SEQ ID NO: 26) survived treatment and grew robustly.
  • Genomic DNA was harvested 3 and 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control;
  • FIG. 4 shows selection for short in-frame deletions in the coding sequence surrounding Q118 and N129 of ATP1A1 following DNA cleavage driven by nuclease G2 (sgRNA G2 + SpCas9) expression and ouabain selection.
  • K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus were transfected with the G2 sgRNA (SEQ ID NO: 24) expression vector and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection. Genomic DNA was harvested from these cells and the TIDE (Tracking of Indels by Decomposition) assay was performed to determine the spectrum and frequency of targeted mutations generated in the pool of cells;
  • TIDE Track of Indels by Decomposition
  • FIG. 5 shows DNA cleavage driven by nuclease G3 (sgRNA G3 + spCas9) within the intron downstream of Q118 and N129 of ATP1A1 does not confer ouabain resistance nor selects for specific insertions or deletions.
  • K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus were transfected with the G3 sgRNA (SEQ ID NO: 25) expression vector and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post- transfection. Genomic DNA was harvested from untreated cells and the TIDE (Tracking of Indels by Decomposition) assay was performed to determine the spectrum and frequency of targeted mutations generated in the pool of cells;
  • FIG. 6 shows selection for short in-frame deletions in the coding sequence surrounding Q118 and N129 of ATP1A1 following DNA cleavage driven by nuclease G4 expression and ouabain selection.
  • K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus were transfected with the G4 sgRNA (SEQ ID NO: 26) expression vector and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection. Genomic DNA was harvested from these cells and the TIDE (Tracking of Indels by Decomposition) assay was performed to determine the spectrum and frequency of targeted mutations generated in the pool of cells;
  • FIG. 7 shows partial amino acid sequences of mutant ATP1A1 polypeptides (SEQ ID NO: 3 (wild-type), SEQ ID NO: 8 ( ⁇ 3), SEQ ID NO: 10 ( ⁇ 6), SEQ ID NO: 12 ( ⁇ 9), SEQ ID NO: 14 ( ⁇ 12) and SEQ ID NO: 16 (A3b)) generated following nuclease treatment and ouabain selection.
  • the region from amino acids S116 to N129 is shown.
  • the indicated single-guide RNA (sgRNA G2 and G4, SEQ ID NOs: 24 and 26) expression vectors (500 ng) were transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus. Cells were treated with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection. Genomic DNA was harvested 10 days post- transfection and the region surrounding the cleavage site was amplified by PCR, TOPO cloned and sequenced;
  • FIG. 8 shows selection for larger in-frame deletions in the coding sequence surrounding Q118 and N129 of ATP1A1 following DNA cleavage driven by nuclease G2 (sgRNA G2 +spCas9) expression and high-dose ouabain selection.
  • K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus were transfected with the G2 sgRNA (SEQ ID NO: 24) expression vector and treated or not with increasing doses of ouabain for 7 days starting 3 days post-transfection. Genomic DNA was harvested from these cells and the TIDE (Tracking of Indels by Decomposition) assay was performed to determine the spectrum and frequency of targeted mutations generated in the pool of cells;
  • TIDE Track of Indels by Decomposition
  • FIG. 9 shows selection for larger in-frame deletions in the coding sequence surrounding Q118 and N129 of ATP1A1 following DNA cleavage driven by nuclease G4 expression and high-dose ouabain selection.
  • K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus were transfected with the G4 sgRNA (SEQ ID NO: 26) expression vector and treated or not with increasing doses of ouabain for 7 days starting 3 days post- transfection. Genomic DNA was harvested from these cells and the TIDE (Tracking of Indels by Decomposition) assay was performed to determine the spectrum and frequency of targeted mutations generated in the pool of cells;
  • FIG. 10 shows amino acid sequences of mutant ATP1A1 polypeptides (SEQ ID NO: 3 (wild-type), SEQ ID NO: 18 ( ⁇ 15), SEQ ID NO: 20 ( ⁇ 18), SEQ ID NO: 16 (A3b), and SEQ ID NO: 22 ( ⁇ 21)) generated following nuclease treatment and high-dose ouabain selection.
  • SEQ ID NO: 3 wild-type
  • SEQ ID NO: 18 ⁇ 15
  • SEQ ID NO: 20 ⁇ 18
  • SEQ ID NO: 16 A3b
  • SEQ ID NO: 22 ⁇ 21
  • the indicated single-guide RNA (sgRNA G2 (A) and G4 (B), SEQ ID NOs: 24 and 26) expression vectors (500 ng) were transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus. Cells were treated with 10 ⁇ ouabain for 7 days starting 3 days post-transfection. Genomic DNA was harvested 10 days post-transfection and the region surrounding the cleavage site was amplified by PCR, TOPO cloned and sequenced;
  • FIGs. 11A-B show that active nucleases targeting the coding sequence surrounding Q118 and N129 of ATP1A1 induce cellular resistance to ouabain.
  • U20S cells were transfected with a wild-type SpCas9 expression vector (500ng) and the indicated sgRNAs (G2-G4 (SEQ ID NOs: 24-26); 500ng). Only cells transfected with sgRNA G2 survived treatment and grew robustly. Genomic DNA was harvested 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • hTERT-RPE-1 cells were transfected with eSpCas9(1.1)_No_FLAG vectors expressing ATP1A1 sgRNAs (G2 (i) or G3 (ii): 500ng) along with ssODN RD (1 ul of a 10 ⁇ stock, (SEQ ID NO: 44)) where indicated. Cells were treated as in (A) and the frequency of mutated alleles was determined using the TIDE assay.
  • RFLP Clal restriction fragment length polymorphism
  • FIG. 12 shows enrichment of cells with targeted mutagenesis at the AAVS1 locus by co-editing the ATP1A1 gene.
  • K562 cells were transfected with two sgRNAs that target respectively ATP1A1 (G2 (SEQ ID NO: 24): 5ng, 50ng, 500ng, panel (A)) and AAVS1 (T2, SEQ ID NO: 468), 5ng and 50ng, panel (B)), in combination with a wild-type SpCas9 expression vector, and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post- transfection.
  • Genomic DNA was harvested 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control.
  • ATP1A1 targeted cells selected by resistance to ouabain show an increased level of indels at the AAVS1 site;
  • FIG. 13 shows enrichment of cells with targeted mutagenesis at the HPRT1 locus by co-editing the ATP1A1 gene.
  • K562 cells were transfected with two sgRNAs that target respectively ATP1A1 (G2 (SEQ ID NO: 24): 5ng, 50ng, 500ng, panel (A)) and HPRT1 -S2L (SEQ IS NO: 456) 5ng and 50ng, panel (B)), in combination with a wild-type SpCas9 expression vector, and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post transfection.
  • G2 SEQ ID NO: 24
  • HPRT1 -S2L SEQ IS NO: 456
  • Genomic DNA was harvested 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control.
  • ATP1A1 targeted cells selected by resistance to ouabain show an increased level of indels at the HPRT1 site;
  • FIG. 14 shows a schematic representation of the intronic SpCas9 target site G3 (Target 3, SEQ ID NO: 29) and partial sequence of single-stranded oligodeoxynucleotides (ssODNs, RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43)) donors used to introduce the Q118D/R and N129D/R mutations conferring ouabain resistance.
  • ssODNs, RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43) partial sequence of single-stranded oligodeoxynucleotides
  • A Wild-type ATP1A1 nucleic acid and amino acid sequences (see SEQ ID NOs: 2 and 3;
  • B Mutation introduced by ssODNs, RD (SEQ ID NO: 44) and
  • C mutation introduced by ssODN DR (SEQ ID NO: 43);
  • FIG. 15 shows the introduction of the Q118R/D and N129D/R mutations via nuclease G3-driven homology-directed repair (HDR) confer cellular resistance to ouabain.
  • the G3 sgRNA (SEQ ID NO: 25) expression vector (500 ng) was co-transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus along with the indicated ssODNs (RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43)). Cells were treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection.
  • Genomic DNA was harvested 3 and 10 days post-transfection and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control;
  • FIG. 16 shows that ouabain selection increases the levels of nuclease-driven HDR at the ATP1A1 locus.
  • the G3 sgRNA (SEQ ID NO: 25) expression vector 500 ng was co-transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus along with the indicated ssODNs (RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43)). Cells were treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post transfection. Only cells co-transfected with sgRNA G3 and ssODNs survived treatment and grew robustly.
  • Genomic DNA was harvested 3 and 10 days post-transfection and a Clal restriction fragment length polymorphism (RFLP) assay was used to determine the frequency of SpCas9-induced HDR at the cleavage site indicated as the % HDR at the base of each lane.
  • An expression vector encoding EGFP (500ng) was used as a negative control;
  • FIG. 17 shows that ouabain selection increases the levels of nuclease-driven HDR at the ATP1A1 locus.
  • the G3 sgRNA expression vector 500 ng was co-transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus along with the indicated ssODNs (RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43)). Cells were treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection. Only cells co- transfected with sgRNA G3 (SEQ ID NO: 25) and ssODNs survived treatment and grew robustly.
  • FIG. 18 shows that ouabain selection increases the levels of nuclease-driven HDR at the ATP1A1 locus.
  • the G3 sgRNA expression vector 500 ng was co-transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus along with the indicated ssODNs RD (SEQ ID NO: 44) and DR (SEQ ID NO: 43)).
  • SEQ ID NO: 44 ssODNs RD
  • DR SEQ ID NO: 43
  • Genomic DNA was harvested 3 and 10 days post-transfection and a Hpy188l restriction fragment length polymorphism (RFLP) assay was used to detect SpCas9-induced HDR of the Q118R mutation.
  • RFLP restriction fragment length polymorphism
  • FIG. 19 shows enrichment of cells with targeted insertion of a point mutation at the HPRT1 locus by co- editing the ATP1A1 gene.
  • K562 cells were transfected with a wild-type SpCas9 expression vector, two sgRNAs that target respectively ATP1A1 (G3 (SEQ ID NO: 25): 50ng and 500ng) and HPRT1-S1R (SEQ ID NO: 465, 50ng) along with ssODNs RD (SEQ ID NO: 44) and a plasmid donor containing homology arms to the HPRT1 locus and a mutation introducing a BamHI restriction site. Cells were treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post transfection.
  • Genomic DNA was harvested 10 days post-transfection and a BamHI restriction fragment length polymorphism (RFLP) assay was used to determine the frequency of SpCas9-induced HDR of the point mutation.
  • RFLP restriction fragment length polymorphism
  • FIG. 20 shows enrichment of cells with targeted integration of a PGK-EGFP expression cassette at the HPRT1 locus by co-editing the ATP1A1 gene.
  • K562 cells were transfected with a wild-type SpCas9 expression vector, two sgRNAs that target respectively ATP1A1 (G3 (SEQ ID NO: 25): 50ng and 500ng) and HPRT1-S1R (SEQ ID NO: 465, 50ng) along with ssODNs RD (SEQ ID NO: 44) and a plasmid donor containing a PGK-EGFP cassette and homology arms to the HPRT1 locus.
  • Cells were treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection. Flow cytometry was used to determine the % of EGFP positive cells in each population;
  • FIG. 21 shows enrichment of cells with in-frame insertion of mAG at the HIST1H2BK c-terminus by co- editing the ATP1A1 gene.
  • K562 cells were transfected with a wild-type SpCas9 expression vector, two sgRNAs that target respectively ATP1A1 (G3 (SEQ ID NOs: 25 (sgRNA) and 29 (target)): (500ng) and HIST1H2BK (SEQ ID NOs: 467 (sgRNA) and 457 (target)) (500ng) along with ssODNs RD (SEQ ID NO: 44) and a plasmid donor containing a mAG cassette and homology arms to the HIST1H2BK locus (donor sequence SEQ ID NO: 474).
  • FIG. 22 shows the enrichment of cells with targeted mutagenesis at the AAVS1 and HPRT1 loci by co- editing the ATP1A1 gene via NHEJ (A) K562 cells stably expressing wild-type SpCas9 were transfected with two sgRNAs that target respectively ATP1A1 (G2 (SEQ ID NOs: 24 (sgRNA) and 28 (target)): 5ng, 50ng, 500ng) and AAVS1 (T2 (SEQ ID NOs: 468 (target) and 469 (sgRNA)): 5ng) and treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection.
  • G2 SEQ ID NOs: 24 (sgRNA) and 28 (target)
  • T2 SEQ ID NOs: 468 (target) and 469 (sgRNA)
  • B Same as in (A) but using the HPRT1 -S2L sgRNA (SEQ ID NO: 466). See also FIGs. 12 and 13 above;
  • FIG. 23 shows a robust enrichment of cells with targeted integrations at endogenous loci by co-editing the ATP1A1 gene via HDR.
  • K562 cells were transfected with combinations of wild-type SpCas9 expression vector (500ng), sgRNAs that target ATP1A1 (G3 (SEQ ID NO: 25): 50ng and 500ng) and HPRT1 (S1 R (SEQ ID NO: 465): 50ng), ssODN RD ((SEQ ID NO: 44), 1 ul of a 10 ⁇ stock) and a plasmid donor containing a PGK-EGFP expression cassette (1 ug) and homology arms to the HPRT1 locus (SEQ ID NO: 480) (cells were transfected with (i) PGK-EGFP donor; (ii) PGK-EGFP donor, HPRT1 sgRNA and ssODN RD; (iii) PGK-EGFP donor, HPRT1 s
  • Cells were treated ((v) to (vii)) or not ((i) to (iv)) with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection.
  • Flow cytometry diagram displaying relative fluorescence intensity on the x- axis and the number of events on the y-axis.
  • K562 cells were transfected with eSpCas9(1.1)_No_FLAG expressing AAVS1- 2 sgRNA (SEQ ID NO: 469,100ng), a pUC19-based ATP1A1 sgRNA vector (G3 (SEQ ID NO: 25): 100ng) along with ssODN RD ((SEQ ID NO: 44) 0.5ul of a 10 ⁇ stock) and a splicing acceptor-SA-2A-EGFP- pA donor (SEQ ID NO: 472) containing homology arms to AAVS1 (250ng) (cells transfected with (i) eSpCas9(1.1); (ii) eSpCas9(1.1), AAVS1- 2 sgRNA, ssODN RD, SA-2A-EGFP and ATP1A1 sgRNA G3; (iii) same as (ii) but cells were treated 0.5 ⁇ ouabain for 7 days starting
  • K562 cells were transfected with a pX330-based vector expressing the LMNA sgRNA (G2 (SEQ ID NO: 471 and 470 (target sequence)): 100 ng), a pUC19-based ATP1A1 sgRNA vector (G3 (SEQ ID NO: 25): 100 ng), along with ssODN RD (0.5ul of a 10 ⁇ stock) and a plasmid donor containing a Clover cassette (250ng) and homology arms to the LMNA locus (SEQ ID NO: 473). Cells were treated (ii) or not (i) with ouabain (0.5 ⁇ ) and analyzed as in (A);
  • FIG. 24 shows enrichment of cells with targeted integrations at endogenous loci by co-editing the ATP1A1 gene via HDR.
  • K562 cells were transfected with a pX330-based vector expressing the LMNA sgRNA (G2: 100 ng, SEQ ID NOs: 470 (target) and 471 (sgRNA)), a pUC19-based ATP1A1 sgRNA vector (G3: 100 ng, (SEQ ID NO: 25)), along with ssODNs RD (0.5ul of a 10 ⁇ stock) and a plasmid donor containing a Clover cassette (250ng, SEQ ID NO: 473) and homology arms to the LMNA locus.
  • G2 100 ng, SEQ ID NOs: 470 (target) and 471 (sgRNA)
  • a pUC19-based ATP1A1 sgRNA vector G3: 100 ng, (SEQ ID NO: 25)
  • FIG. 20 Cells were treated as in FIG. 20 (treated or not with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection) and flow cytometry was used to determine the % of Clover positive cells in each population.
  • B Exemplary fluorescence imaging of ouabain treated cells expressing the Clover-LMNA fusion. See also FIG. 23C;
  • FIG. 25 shows that high specificity CRISPR nucleases AsCpfl and eSpCas9 (1.1) are compatible with the ouabain selection strategy.
  • A Schematic representation of AsCpfl target sites surrounding DNA encoding the first extracellular loop of human ATP1A1. Annotated are the positions of residues Q118 and N129, exon/intron boundary, protospacer adjacent motifs (PAM) and five potential AsCpfl target sequences (Targets 1-5, SEQ ID NOs: 37-41).
  • the indicated crRNA expression vectors (G5-G9 SEQ ID NOs: 32-36 (RNA sequence) (500 ng) were transfected into K562 cells stably expressing AsCpfl from the AAVS1 safe harbor locus. Genomic DNA was harvested 10 days post-transfection and the Surveyor assay was used to determine the frequency of AsCpfl - induced insertions and deletions indicated as the % Indels at the base of each lane. Where indicated cells were treated with 0.5 ⁇ ouabain for 7 days starting 3 days post transfection. An expression vector encoding EGFP (-) was used as a negative control.
  • FIG. 26 shows the enrichment of NHEJ- and HDR-driven events in primary human cord blood (CB) CD34+ cells upon selection with ouabain.
  • A Cultured CD34+ cells (100 000-200 000 per sample) were electroporated with Cas9 RNP G2 and grown for the indicated period of time in presence of 0.5 ⁇ ouabain starting 6 days post-transfection. Genomic DNA was harvested and the Surveyor assay was used to determine the frequency of SpCas9-induced insertions and deletions indicated as the % Indels at the base of each lane. Recombinant Cas9 (-) was used as a negative control.
  • B The region surrounding the cleavage site was amplified by PCR and analyzed by TIDE assay.
  • CD34+ cells were treated as in (A) but ssODN RD (1 ul of a 50 ⁇ stock) was added to the transfection mixes.
  • a BmgBI restriction fragment length polymorphism (RFLP) assay was used to determine the frequency of SpCas9-induced HDR at the cleavage site indicated as the % HDR at the base of each lane;
  • FIG. 27 shows the strategy for CRISPR/Cas9-driven HDR at the MGMT locus to protect hematopoietic cells from the combination of 06-benzylguanine and BCNU.
  • SEQ ID NO: 484 Annotated are the positions of residues P140 and G156, exon/intron boundary and protospacer adjacent motifs (PAM).
  • PAM protospacer adjacent motifs
  • FIGs. 28A-I show non-limiting examples of SpCas9 target sequences and their associated PAM for ATP1A.
  • A Exon 5 (SEQ ID NOs: 90-123);
  • B Exon 8 (SEQ ID NOs: 124-182);
  • C Exon 9 (SEQ ID NOs: 183-218);
  • D Exon 17 (SEQ ID NOs: 219-252); Exon 18 (SEQ ID NOs:253-293);
  • F-G Exons 19-20 (SEQ ID NOs: 294-381 ;
  • H Exon 21 (SEQ ID NOs: 382-414); and
  • I Exon 22 (SEQ ID NOs: 415-448);
  • FIG. 29 shows enrichment of cells with in-frame insertion of mAG at the HIST1H2BK c-terminus by co- editing the ATP1A1 gene under increasing doses of ouabain.
  • K562 cells were transfected with eSpCas9(1.1)_No_Flag_ATP1A1_G3_dual_H2BK (100ng), a vector that drives the expression of eSpCas9(1.1) along with two sgRNAs that target respectively ATP1A1 and HIST1H2BK (SEQ ID NOs: 29/25 and 457/467).
  • the transfection mix was supplemented with ssODNs RD (5pmol) and a plasmid donor containing a mAG cassette and homology arms to the HIST1H2BK locus (250ng, SEQ ID NO: 474).
  • Cells were treated with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection, and then the dose was increased to 100 ⁇ for another seven days. Finally, cells selected with 100 ⁇ ouabain were exposed to 1000 ⁇ for another 7 days. Flow cytometry was used to determine the % of mAG positive cells in each population;
  • FIG. 30 shows a schematic of the strategy for targeted parallel integration of an exogenous sequence into the ATP1A1 locus.
  • Targeted integration of the exogenous sequence occurs within intron 4 (the polynucleotide sequence of intron 4 corresponds to nts 305-404 of SEQ ID NO: 2), in proximity to exon 4 (the polynucleotide sequence of exon 4 corresponds to nts 101-304 of SEQ ID NO: 2) that encodes Q118 and N129 (vertical black bars).
  • the positions of the DNA double-strand break (DSB) generated within intron 4 by either SpCas9 (G3- SEQ ID NO: 29 (target sequence)) or AsCpfl (G9-SEQ ID NO: 41 (target sequence)) are predicted to be 19bp and 49bp downstream of exon 4, respectively.
  • the donor DNA molecule directs the integration of an expression cassette encoding a fluorescent protein (FP) in the same direction as the ATP1A1 transcript (parallel orientation).
  • the left homology arm (L-HA) of the donor DNA molecule contains the Q118R and N129D (RD) mutations (*asterisks).
  • hPGKf human phosphoglycerate kinase 1 gene promoter, bGHpA: polyadenylation signal from the bovine growth hormone gene;
  • FIG. 31 shows a schematic of the strategy for targeted antiparallel integration of an exogenous sequence into the ATP1A1 locus.
  • Targeted integration of the exogenous sequence occurs within intron 4 (the polynucleotide sequence of intron 4 corresponds to nts 305-404 of SEQ ID NO: 2), in proximity to exon 4 (the polynucleotide sequence of exon 4 corresponds to nts 101-304 of SEQ ID NO: 2) that encodes Q118 and N129 (vertical black bars).
  • the positions of the DNA double-strand break (DSB) generated within intron 4 by either SpCas9 (G3- SEQ ID NO: 29 (target sequence)) or AsCpfl (G9-SEQ ID NO: 41 (target sequence)) are predicted to be 19bp and 49bp downstream of exon 4, respectively.
  • the donor DNA molecule directs the integration of an expression cassette encoding a fluorescent protein (FP) in opposite direction to the ATP1A1 transcript (antiparallel orientation).
  • the left homology arm (L-HA) of the donor DNA molecule contains the Q118R and N129D (RD) mutations (*asterisks).
  • hPGKf human phosphoglycerate kinase 1 gene promoter, bGHpA: polyadenylation signal from the bovine growth hormone gene;
  • FIG. 32 shows the expression level of the TurboGFP protein (GFP-A) upon targeted integration of the expression cassette in the parallel (A) versus antiparallel (B) orientation as detailed in FIGs. 30-31.
  • K562 cells were transfected with eSpCas9(1.1)_No_Flag_ATP1A1_G3 (500ng), a vector that drives the expression of eSpCas9(1.1) along with the sgRNA that targets intron 4 of ATP1A1 (G3 -SEQ ID NOs:25 (sgRNA) and 29 (target)).
  • the transfection mix was supplemented with the plasmid donors (500ng, SEQ ID NOs: 475 (parallel) and 476 (antiparallel), as indicated.
  • Cells were left untreated or were treated with 0.5 ⁇ ouabain for 10 days starting 3 days post-transfection. Flow cytometry was used to determine the % of TurboGFP positive cells in each population;
  • FIG. 33 shows the expression level of the mNeonGreen protein upon targeted integration of the expression cassette in the antiparallel orientation as directed by two donor molecules (v1 (A, SEQ ID NO: 477) and v2 (B, SEQ ID NO: 478)) differing by the extent of homology present at the 3' end of the left homology arm.
  • the left homology arm of donor molecule v1 spans and extends 27bp 3' of the predicted DSB.
  • the left homology arm of donor molecule v2 spans and extends 6bp 3' of the predicted DSB. Otherwise, the structure of the donors corresponds to FIGs. 30-31.
  • K562 cells were transfected with eSpCas9(1.1)_No_Flag_ATP1A1_G3 (500ng), a vector that drives the expression of eSpCas9(1.1) along with the sgRNA that targets intron 4 of ATP1A1.
  • the transfection mix was supplemented with the plasmid donors (1000ng), as indicated.
  • Cells were left untreated or were treated with 0.5 ⁇ ouabain for 10 days starting 3 days post-transfection. Flow cytometry was used to determine the % of mNeonGreen positive cells in each population;
  • FIG. 34 shows the expression level of the mNeonGreen protein upon targeted integration of the expression cassette in the antiparallel orientation as directed by donor molecule v2 (see description of FIG. 33 above) and either the eSpCas9 (G3, SEQ ID NOs: 25 (sgRNA) and 29 (target sequence)) or AsCpfl (G9, SEQ ID NO: 36 (sgRNA) and 41 (target)) nucleases.
  • K562 cells were transfected with either eSpCas9(1.1)_No_Flag_ATP1A1_G3 (500ng) or pY036_ATP1A1_G9 (500ng), vectors that drive the expression of eSpCas9(1.1) or AsCpfl along with the sgRNA/crRNA that targets intron 4 of ATP1A1.
  • the transfection mix was supplemented with the plasmid donor (1000ng), as indicated.
  • Cells were left untreated or were treated with 0.5 ⁇ ouabain for 10 days starting 3 days post-transfection. Flow cytometry was used to determine the % of mNeonGreen positive cells in each population;
  • FIG. 35 shows the expression level of the mNeonGreen protein upon targeted integration of the expression cassette in the antiparallel orientation as directed by donor molecule v2 and the AsCpfl (G9) nucleases.
  • K562 cells were transfected with pY036_ATP1A1_G9 (SEQ ID NOs: 36/41 , 500ng), a vector that drives the expression of AsCpfl along with the crRNA that targets intron 4 of ATP1A1 (see SEQ ID NO: 2).
  • the transfection mix was supplemented with the plasmid donor at two different doses (500ng (A) and 1000ng (B)), as indicated.
  • Cells were left untreated or were treated with 0.5 ⁇ ouabain for 10 days starting 3 days post-transfection. Flow cytometry was used to determine the % of mNeonGreen positive cells in each population;
  • FIG. 36 shows enrichment of cells with targeted integrations at the endogenous LMNA locus by co- editing the ATP1A1 gene via homology-driven targeted integration of an exogenous expression cassette encoding a fluorescent protein (FP).
  • K562 cells were transfected with eSpCas9(1.1)_No_Flag_ATP1A1_G3_dual_LMNA (100ng), a vector that drives the expression of eSpCas9(1.1) along with two sgRNAs that target respectively ATP1A1 and LMNA (G3 (SEQ ID NOs: 23 (target) and 29 (sgRNA) and G2 (SEQ ID NOs:470 (target) and 471 (sgRNA)).
  • the transfection mix was supplemented with plasmid donors containing an mScarlet (red fluorescent protein) cassette and homology arms to the LMNA locus (250ng, SEQ ID NO: 479) in addition to the ATP1A1 mNeonGreen antiparallel v1 donor (see FIG. 33 and Examples 8 and 9, SEQ ID NO: 477) 250ng) (See FIGs 31-32).
  • Cells were left untreated or were treated with 0.5 ⁇ ouabain for 10 days starting 3 days post-transfection. Flow cytometry was used to determine the % of mScarlet and mNeonGreen positive cells in each population; and
  • FIGs. 37A-B show a protein sequence alignment between ATP1A1 (SEQ ID NO: 3), ATP1A2 SEQ ID NO: 460), ATP 1 A3 SEQ ID NO: 462) and ATP1A4 (SEQ ID NO: 464).
  • A Alignment of amino acids 1-598 of ATP1A1. Exon 4 region (aa 124-138-) is shown between lines;
  • B Alignment of amino acids 599-1023 of ATP1A1.
  • Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing editing efficiencies in a wide variety of biomedically important cell types would facilitate the analysis of gene function. Harnessing the multiplexing capabilities of the clustered, regularly interspaced, short palindromic repeat (CRISPR) system, described herein is a robust co-selection strategy enabling enrichment of cells harboring nuclease-driven non-homologous end joining (NHEJ) and homology-directed repair (HDR) events.
  • CRISPR clustered, regularly interspaced, short palindromic repeat
  • An aspect of the system disclosed herein is that the co-selection process can be initiated through NHEJ or HDR processes independently.
  • One only has to switch between sgRNAs targeting juxtaposed regions of gene e.g., in an intron of an endogenous gene locus conferring resistance to a drug such as ATP1A1 and include a donor or patch nucleic acid (e.g., ssODN) to co-select for HDR- instead of NHEJ-driven events.
  • Methods of the present invention are expected to be compatible with any engineered nuclease platforms and any cell type when ubiquitously expressed genes (e.g., ATP1A1) are used.
  • the potent and rapid action of the drug combined with the ability to increase the frequency of HDR or NHEJ-driven modification(s) facilitates the incorporation of (e.g., marker- free) genetic changes in human cells while avoiding extensive screening and optimization. Furthermore, the absence of use of exogenous markers is advantageous in the case of therapeutic applications.
  • Described herein is a method that can be broadly applied to increase the frequency of genome editing events.
  • the method comprises creating and selecting for dominant gain-of-function alleles of endogenous genes using a selected protein/drug combination.
  • the modified protein/drug combination can then effectively be used to enrich for custom modifications at unlinked loci of interest.
  • methods comprising creating a mutated form of an endogenous gene which in turn encodes a mutated form of the protein that is more resistant to a corresponding drug.
  • the mutated form of the protein is more resistant to a corresponding protein lacking the mutation, which is less resistant/more sensitive to the drug.
  • the mutation(s) or modification(s) in the endogenous gene and in turn the encoded protein confer resistance or greater resistance to the drug, relative to a corresponding form of the protein lacking the mutation(s) or modification(s).
  • Non-limiting examples of useful protein/drug combinations are listed in Table 1. Note that several combinations of mutations can confer resistance to a given drug, and thus other possible mutations or mutation combinations can be identified. Specific combinations may be selected for use in a tissue/cell type specific manner.
  • Table 1 Example of mutants of endogenous human enzymes resistant to cytotoxic drugs that could be used for in vitro or in vivo co-selection.
  • ATP1A4 480 ATP1A4 : HGNC: 14073;
  • co-enrichment of edited cells either in vitro or in vivo may be achieved for example after chemo-selection of cells modified to express the P140K and/or G156A variants of human 0(6)-methylguanine-DNA-methyltransferase (MGMT wild type sequence: NM_002412.4 (SEQ ID NO: 481), NP_002403.2 (SEQ ID NO: 482); see FIG. 27).
  • MGMT wild type sequence: NM_002412.4 SEQ ID NO: 481
  • NP_002403.2 SEQ ID NO: 482
  • MGMT repairs DNA damage by removing adducts from the 06 position of guanine, decreasing the cytotoxic effects of alkylating agents such as dacarbazine, temozolomide (TMZ), procarbazine, and nitrosoureas such as 1 ,3-bis-(2-chloroethyl)-1 - nitrosourea (BCNU).
  • alkylating agents such as dacarbazine, temozolomide (TMZ), procarbazine, and nitrosoureas such as 1 ,3-bis-(2-chloroethyl)-1 - nitrosourea (BCNU).
  • BG 06-benzylguanine
  • BG 06-benzylguanine
  • these residues are encoded next to an intron-exon boundary and could be targeted in a similar fashion as demonstrated herein for ATP1 A1 to favor enrichment of cells edited via HDR (see FIG. 27).
  • DHFR/methotrexate Another example of protein/drug combination that may be used in accordance with the present invention is DHFR/methotrexate. Protection of cells during administration of antifolate chemotherapy, such as methotrexate (MTX) and trimetrexate (TMTX), can be achieved by overexpression of a mutant form of the DHFR enzyme (DHFR L22Y) that is resistant to MTX inhibition. Co-selection could be achieved through introduction of the L22Y mutation at the endogenous DHFR gene via HDR.
  • MTX methotrexate
  • TTTX trimetrexate
  • IMPDH2 inosine monophosphate dehydrogenase 2
  • MP A mycophenolic acid
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps and are used interchangeably with the phrase “including but not limited to”.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001 ; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P. M. Wassarman and A. P.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double- stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double- stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.
  • non-conservative mutation or “non-conservative substitution” in the context of polypeptides refers to a mutation in a polypeptide that changes an amino acid to a different amino acid with different biochemical properties (i.e., charge, hydrophobicity and/or size).
  • a non-conservative substitution includes one that changes an amino acid of one group with another amino acid of another group (e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
  • an amino acid of one group e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
  • a "conservative substitution” or “conservative mutations” in the context of polypeptides are mutations that change an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size).
  • a leucine and isoleucine are both aliphatic, branched hydrophobes.
  • aspartic acid and glutamic acid are both small, negatively charged residues. Therefore, changing a leucine for an isoleucine (or vice versa) or changing an aspartic acid for a glutamic acid (or vice versa) are examples of conservative substitutions.
  • Coding sequence or "encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein or sgRNA.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.
  • the coding sequence may be codon optimized.
  • “Complement” or “complementary” as used herein refers to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.
  • Homology and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences.
  • nucleic acid sequence is "substantially homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term “homologous” does not infer evolutionary relatedness, but rather refers to substantial sequence identity, and thus is interchangeable with the terms “identityTidentical”).
  • Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs.
  • sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
  • the units e.g., 66, 67...81 , 82,...91, 92%.
  • Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. (Altschul et al. 1990) 1990 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/).
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.
  • hybridize to each other under moderately stringent, or preferably stringent, conditions Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1 % SDS at 42°C (Ausubel 2010).
  • hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1 % SDS at 68°C (Ausubel 2010).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (Tijssen 1993).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • Binding refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid or between a sgRNA and a target polynucleotide or between a sgRNA and a CRISPR nuclease (e.g., Cas9, Cpf1). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.
  • Affinity refers to the strength of binding: increased binding affinity being correlated with a lower Kd.
  • a "binding protein” is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a DNA-binding protein a DNA-binding protein
  • an RNA-binding protein an RNA-binding protein
  • a protein-binding protein it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA- binding, RNA-binding and protein-binding activity.
  • Ouabain (Cas No: 630-60-4), also known as g-strophanthin, is a cardiac glycoside that inhibits Na+/K+- ATPase sodium-potassium ion pump. It is a plant derived toxic substance that was traditionally used as an arrow poison in eastern Africa for both hunting and warfare. Ouabain is a cardiac glycoside and in lower doses, can be used medically to treat hypotension and some arrhythmias. It acts by inhibiting the Na+/K-i-ATPase sodium- potassium ion pump. Once ouabain binds to this enzyme, the enzyme ceases to function, leading to an increase of intracellular sodium.
  • NCX sodium-calcium exchanger
  • the term "ouabain derivative” or “ouabain-like agen refers to an agent which, like ouabain, binds to the ATP1A1 polypeptide, inhibits the Na+/K-i-ATPase sodium-potassium ion pump and is toxic to cells (may kill cells at a certain concentration). It includes certain cardiotonic steroids (CTS) which may be sufficiently toxic to cells to enable proper selection of target cells in accordance with methods of the present invention.
  • CTS cardiotonic steroids
  • the term "ouabain resistance” refers to cellular resistance (as compared to normal cells, e.g., cells comprising an ATP1A1 gene lacking the modification conferring the ouabain resistance) to ouabain and/or ouabain derivatives. Such resistant cells are also referred to herein as being tolerant to a toxin such as a cardiotonic steroid (CTS), e.g., ouabain and/or a ouabain derivative. "Tolerance” or “tolerant” as used herein refers to the capacity of a first cell to be less affected by a toxin than a second cell that does not have a modified ATP1A1 gene that confers resistant to the toxin.
  • CTS cardiotonic steroid
  • Tolerant cells grow and develop better in the presence of a toxin when compared to intolerant cells, i.e. have increased growth and viability relative to intolerant cells.
  • Such tolerance can be used as a basis of selection of tolerant cells over intolerant cells, e.g. based on increased growth of tolerant cells relative to intolerant cells when treated with the toxin, in an embodiment the viability of tolerant cells relative to the viability of intolerant cells when treated with the toxin.
  • a nuclease-based modification refers to a modification in a polynucleotide e.g., an endogenous gene locus or genomic sequence) which involves the introduction of a cut (e.g., a double-stranded break in the polynucleotide) which ultimately will trigger a repair mechanism by the cell involving (Non-homologous- end-joining) NHEJ or homologous recombination (HDR).
  • the nuclease-based modification is made by site specific nucleases targeting the polynucleotide of interest (i.e., an endogenous gene locus or genomic sequence).
  • Zinc finger DNA binding protein (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • a "TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
  • a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in 55 length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, also, U.S. Patent Publication No. 20110301073.
  • Zinc finger binding domains can be "engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein.
  • TALEs can be “engineered” to bind to a predetermined nucleotide sequence, for example by engineering of the amino acids involved in DNA binding (the "Repeat Variable Diresidue” or "RVD” region). Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non naturally occurring.
  • Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design/composition results principally from rational criteria.
  • Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081 ; 6,453,242; and 6,534, 261 ; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. application Ser. No. 13/068,735.
  • Recombination refers to a process of exchange of genetic information between two polynucleotides.
  • HR homologous recombination
  • HDR homology-directed repair
  • This process requires nucleotide sequence homology, uses a "donor” molecule as a template for repair of a "target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis- dependent strand annealing," in which the donor is used to re-synthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • one or more targeted (site-specific) nucleases create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site.
  • a "donor" polynucleotide having homology to the nucleotide sequence in the region of the break, may be introduced into the cell if desired (e.g., to introduce a specific modification in the ATP1A1 gene to generate a ouabain resistant ATP1A1 polypeptide and into one or more further target genes to introduce one or more further desired modifications).
  • the presence of the double-stranded break has been shown to facilitate integration of the donor sequence.
  • the donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin.
  • a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide.
  • the use of the terms "replace” or "replacement” can be understood to represent replacement of one nucleotide sequence by another, (i.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another.
  • additional sgRNA/CRISPR nucleases, pair zinc-finger, Meganucleases, Mega-Tals, and/or additional TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell.
  • the terms "donor” or “patch” nucleic acid are used interchangeably and refers to a nucleic acid that includes a fragment of the endogenous targeted gene of a cell (in some embodiments the entire targeted gene), but which includes desired modification(s) at specific nucleotides (e.g., a fragment of the ATP1A1 gene comprising targeted mutations conferring resistance to cardiotonic steroids (CTS) such as ouabain).
  • CTS cardiotonic steroids
  • the donor (patch) nucleic acid must be of sufficient size and similarity (e.g., in the right and left homology arms) to permit homologous recombination with the targeted gene.
  • the donor/patch nucleic acid is (or is flanked at the 5' end and at the 3' end by sequences) at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the endogenous targeted polynucleotide gene sequence.
  • the patch nucleic acid may be provided for example as a ssODN, as a PCR product (amplicon) or within a vector.
  • the patch/donor nucleic acid will include modifications with respect to the endogenous gene which i) precludes it from being cut by a sgRNA once integrated in the genome of a cell and/or which facilitate the detection of the introduction of the patch nucleic acid by homologous recombination.
  • a "targeted gene” or “targeted polynucleotide” corresponds to the polynucleotide within a cell that will be modified by the introduction of the patch nucleic acid. It corresponds to an endogenous gene naturally present within a cell.
  • the targeted gene may comprise one or more mutations associated with a risk of developing a disease or disorder which may be corrected by the introduction of the patch/donor nucleic acid (e.g., will be modified to correspond to the WT gene or to a form which is no longer associated with increased risk of developing a disease or condition).
  • One or both alleles of a targeted gene may be corrected or modified within a cell in accordance with the present invention.
  • a "target polynucleotide” as used herein refers to any endogenous polynucleotide or nucleic acid present in the genome of a cell and encoding or not a known gene product.
  • “Target gene” as used herein refers to any endogenous polynucleotide or nucleic acid present in the genome of a cell and encoding a known or putative gene product (siRNA, protein, etc.).
  • the target gene or target polynucleotide further corresponds to the polynucleotide within a cell that will be modified by a nuclease of the present invention, alone or in combination with the introduction of one or more donor nucleic acid or patch nucleic acids.
  • the target gene or target polynucleotide may be a mutated gene involved in a genetic disease.
  • Promoter means a synthetic or naturally-derived nucleic acid molecule which is capable of conferring, modulating or controlling (e.g., activating, enhancing and/or repressing) expression of a nucleic acid in a cell.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance or repress expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.
  • the U6 promotor is used to express one or more sgRNAs in a cell.
  • Vector as used herein means a nucleic acid sequence containing an origin of replication.
  • a vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector.
  • a vector may be a self- replicating extrachromosomal vector, and preferably, is a DNA plasmid.
  • the vector may comprise nucleic acid sequence(s) that/which encode(s) a sgRNA, a donor (or patch) nucleic acid, and/or a CRISPR nuclease (e.g., Cas9 or Cpf1) of the present invention.
  • a vector for expressing one or more sgRNA will comprise a "DNA" sequence of the sgRNA.
  • Adeno-associated virus or "AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.
  • Gene editing and selection methods of the present invention allow to rapidly select and enrich for genome edited cells.
  • methods of the present invention further allow to enrich for certain types of modifications (i.e., increase the efficiency of a cell to generate a specific type of modification(s) e.g., knock o u t/d e leti o n/i n acti vati o n vs targeted insertion/mutation(s)) by selectively favoring Homologous directed repair (HDR) or Non-homologous end joining (NHEJ).
  • HDR Homologous directed repair
  • NHEJ Non-homologous end joining
  • Methods of the present invention involve generating a ATP1 A1 polypeptide resistant to cardiotonic steroids (CTS), such as ouabain by cleaving with a nuclease within the endogenous ATP1A1 gene. Ouabain resistance was shown to allow for rapid selection and efficient enrichment of genome-edited cells. The sole introduction of targeted double stranded breaks (DSB) within the APT1A1 gene sequence was also surprisingly found to generate spontaneous deletion mutants/variants resistant to ouabain.
  • CTS cardiotonic steroids
  • Various types of endonucleases or nickases may be used to induce a DSB at selected site(s) in one or more targeted polynucleotides.
  • useful endonucleases and nickases include meganucleases, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector nucleases (TALENs), Mega- Tals, or the CRISPR nucleases or nickases used in combination with at least one (e.g., one or two) guide RNA(s) (sgRNA) in the Clustered regularly interspaced short palindrome repeat (CRISPR) system.
  • sgRNA guide RNA(s)
  • CRISPR Clustered regularly interspaced short palindrome repeat
  • the present invention uses the CRISPR system (i.e., combination of sgRNA and CRISPR nuclease or nickase such as Cas9 and Cpf1), optionally together with a donor (patch) polynucleotide sequence to generate ouabain resistance and introduce one or more genetic modifications in a target endogenous polynucleotide/gene.
  • CRISPR system i.e., combination of sgRNA and CRISPR nuclease or nickase such as Cas9 and Cpf1
  • a donor (patch) polynucleotide sequence to generate ouabain resistance and introduce one or more genetic modifications in a target endogenous polynucleotide/gene.
  • sgRNAs can be used with a CRISPR nuclease to generate ouabain resistance and to increase frequency of specific genome editing events in one or more targeted polynucleotide human cells.
  • CRISPR technology is a system for genome editing, e.g., for modification of the expression of a specific gene.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the Cas9 protein was directed to genomic target sites by a synthetically reconstituted "guide RNA" ("sgRNA”), corresponding to a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. It comprises a “sgRNA guide sequence” or “sgRNA target sequence” and an RNA sequence (Cas recognition sequence)", which is necessary for Cas (e.g., Cas9) binding to the targeted gene.
  • the sgRNA guide sequence is the sequence that confers specificity. It hybridizes with (i.e., it is complementary to) the opposite strand of a target sequence (i.e., it corresponds to the RNA sequence of a DNA target sequence).
  • CRISPR systems using different CRISPR nucleases have been developed and are known in the art (e.g., using the Cpf1 nuclease instead of a Cas9 nuclease).
  • CRISPR/nuclease-based engineered systems for use in modifying the ATP1A1 gene and inducing ouabain resistance in cells.
  • the CRISPR-based engineered systems of the present invention are designed to (i) target and cleave the ATP1A1 gene (e.g., in ATP1A1 intron 3, exon 4 or intron 4) to generate ATP1A1 variants conferring ouabain resistance (deletion or insertion variants (i.e., NHEJ-induced variants) or specifically selected and targeted variants (i.e., HDR-induced variants using a donor ATP1A1 polynucleotide comprising the desired mutation(s)/modification(s)), (ii) increase the frequency of specific genome editing events (i.e., favoring NHEJ or HDR-directed modifications) in cells; and/or (iii) simplify and/or increase the speed of identification and selection of genome-edited target cells having the
  • the CRISPR/nuclease-based systems of the present invention include at least one CRISPR nuclease (e.g. a Cas9 or Cpf1 nuclease) and at least one sgRNA targeting the endogenous ATP1A1 gene in target cells.
  • CRISPR nuclease e.g. a Cas9 or Cpf1 nuclease
  • sgRNA targeting the endogenous ATP1A1 gene in target cells.
  • the present invention involves the design and preparation of one or more sgRNAs for inducing a DSB (or two single stranded breaks (SSB) in the case of a nickase) in ATP1A1.
  • the present invention also involves the design and preparation of one or more sgRNAs for inducing a DSB (or two SSBs in the case of a nickase) in a target polynucleotide located at a different locus within the genome of target cells.
  • the sgRNAs targeting the ATP1A1 gene or targeting the ATP1A1 gene and the target polynucleotide(s)) and the nuclease are then used together to introduce the desired modification(s) (i.e., gene- editing events) by NHEJ or HDR within the genome of one or more target cells.
  • the desired modification(s) include specific point mutation(s) or insertions/deletion(s) (e.g., in the ATP1A1 gene or in the ATP1A1 gene and target polynucleotide(s)
  • one or more donor or patch nucleic acids comprising the desired modification(s) are provided to introduce the modification(s) by HDR.
  • CRISPR nucleases require the presence of a sgRNA and a of a protospacer adjacent motif (PAM) on the targeted gene.
  • PAM protospacer adjacent motif
  • the PAM immediately follows (i.e., is adjacent to) the sgRNA target sequence in the targeted polynucleotide gene sequence.
  • the PAM is located at the 3' end or 5' end of the sgRNA target sequence (depending on the CRISPR nuclease used) but is not included in the sgRNA guide sequence.
  • the PAM for Cas9 CRSIPR nucleases is located at the 3' end of the sgRNA target sequence on the target gene while the PAM for Cpf1 nucleases is located at the 5' end of the sgRNA target sequence on the target gene.
  • Different CRISPR nucleases also require a different PAM. Accordingly, selection of a specific polynucleotide sgRNA target sequence (e.g., on the ATP1A1 gene nucleic acid sequence) is generally based on the CRISPR nuclease used.
  • the PAM for the Streptococcus pyogenes Cas9 CRISPR system is 5 -NRG- 3', where R is either A or G, and characterizes the specificity of this system in human cells.
  • the PAM of S. aureus Cas9 is NNGRR.
  • the S. pyogenes Type II system naturally prefers to use an "NGG" sequence, where "N” can be any nucleotide, but also accepts other PAM sequences, such as "NAG” in engineered systems.
  • the Cas9 derived from Neisseria meningitidis normally has a native PAM of NNNNGATT, but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM.
  • the PAM for AsCpfl or LbCpfl CRISPR nuclease is TTTN.
  • the PAM for a Cas9 protein used in accordance with the present invention is a NGG trinucleotide-sequence (Cas9).
  • the PAM for a Cpf1 CRISPR nuclease used in accordance with the present invention is a TTTN nucleotide sequence. Table 2 below provides a list of non-limiting examples of CRISPR/nuclease systems with their respective PAM sequences.
  • Table 2 Non-exhaustive list of CRISPR-nuclease systems from different species (see. Mohanraju, P. et al., PMID 27493190; Shmakov, S et al., PMID: 26593719; and Zetsche, B. et al., PMID: 26422227). Also included are engineered variants recognizing alternative PAM sequences (see Kleinstiver, BP. et al., (Nature biotech 2015) PMID: 26524662 and Kleinstiver, BP. et al., (Nature 2015)).
  • SA Staphylococcus aureus
  • SaCas9 NNGRRT or NNGRR(N) CRISPR nuclease PAM Sequence
  • sgRNA refers to a guide RNA which works in combination with a CRISPR nuclease to introduce a cut into DNA.
  • the sgRNA comprises a sgRNA guide sequence and a "CRISPR nuclease recognition sequence”.
  • sgRNA guide sequence refers to the corresponding RNA sequence of the "sgRNA target sequence". Therefore, it is the RNA sequence equivalent of the protospacer on the target polynucleotide gene sequence. It does not include the corresponding PAM sequence in the genomic DNA. It is the sequence that confers target specificity.
  • the sgRNA guide sequence is linked to a CRISPR nuclease recognition sequence which binds to the nuclease (e.g., Cas9/Cpf1). The sgRNA guide sequence recognizes and binds to the targeted gene of interest.
  • the PAM is the nucleic acid sequence, that immediately follows (is contiguous to) the target sequence on the ATP1A1 gene or target polynucleotide but is not in the sgRNA.
  • Exemplary PAM and target sequences (corresponding to the DNA sequence of the sgRNA guide sequence) targeting the ATP1A1 gene are shown in Table 3, below.
  • a "CRISPR nuclease recognition sequence” as used herein refers broadly to one or more RNA sequences (or RNA motifs) required for the binding and/or activity (including activation) of the CRISPR nuclease on the target gene. Some CRISPR nucleases require longer RNA sequences than other to function. Also, some CRISPR nucleases require multiple RNA sequences (motifs) to function while others only require a single short RNA sequence/motif. For example, Cas9 proteins require a tracrRNA sequence in addition to a crRNA sequence to function while Cpfl only requires a crRNA sequence (e.g., SEQ ID NO: 41 for AsCpfl).
  • Cpfl processes crRNA arrays independent of tracrRNA, and Cpf1 -crRNA complexes alone cleave target DNA molecules, without the requirement for any additional RNA species (see Zetsche et al., PMID: 26422227).
  • CRISPR nuclease recognition sequence included in the sgRNA described herein is thus selected based on the specific CRISPR nuclease used. It includes direct repeat sequences and any other RNA sequence known to be necessary for the selected CRISPR nuclease binding and/or activity. Various RNA sequences which can be fused to an RNA guide sequence to enable proper functioning of CRISPR nucleases (referred to herein as CRISPR nuclease recognition sequence) are well known in the art and can be used in accordance with the present invention.
  • the "CRISPR nuclease recognition sequence” may thus include a crRNA sequence only (e.g., for AsCpfl activity, such as the CRISPR nuclease recognition sequence UAAUUUCUAC UCUUGUAGAU set forth in SEQ ID NO: 41) or may include additional sequences (e.g., tracrRNA sequence necessary for Cas9 activity, such as the CRISPR nuclease recognition sequence set forth in SEQ ID NO: 31 which includes both crRNA and tracrRNA sequences).
  • a crRNA sequence e.g., for AsCpfl activity, such as the CRISPR nuclease recognition sequence UAAUUUCUAC UCUUGUAGAU set forth in SEQ ID NO: 41
  • additional sequences e.g., tracrRNA sequence necessary for Cas9 activity, such as the CRISPR nuclease recognition sequence set forth in SEQ ID NO: 31 which includes both crRNA and tracrRNA sequences.
  • RNA motifs necessary for CRISPR nuclease binding and activity may be provided separately (e.g., (i) RNA guide sequence- crRNA CRISPR recognition sequence" (also known as crRNA) in one RNA molecule and (ii) a tracrRNA CRISPR recognition sequence on another, separate RNA molecule.
  • RNA guide sequence- crRNA CRISPR recognition sequence also known as crRNA
  • a tracrRNA CRISPR recognition sequence on another, separate RNA molecule e.g., all necessary RNA sequences (motifs) may be fused together in a single RNA guide.
  • the CRISPR recognition sequence is preferably fused directly to the sgRNA guide sequence (in 3' (e.g., Cas9) or 5' (Cpfl) depending on the CRISPR nuclease used) but may include a spacer sequence separating two RNA motifs.
  • the CRISPR nuclease recognition sequence is a Cas9 recognition sequence having at least 65 nucleotides. In embodiments, the CRISPR nuclease recognition sequence is a Cas9 CRISPR nuclease recognition sequence having at least 85 nucleotides. In embodiments, the CRISPR nuclease recognition sequence is a Cpfl recognition sequence (5' direct repeat) having about 19 nucleotides.
  • the Cas9 recognition sequence comprises (or consists of) the sequence as set forth in SEQ ID NO: 31.
  • the AsCpfl recognition sequence comprises (or consists of) the sequence UAAUUUCUAC UCUUGUAGAU (SEQ ID NO: 42).
  • the sgRNA of the present invention may comprise any variant of the above noted sequences, provided that it allows for the proper functioning of the selected CRISPR nuclease (e.g., binding of the CRISPR nuclease protein to the ATP1A1 gene and/or target polynucleotide sequence(s)).
  • RNA guide sequence and CRSIPR nuclease recognition sequence(s) provide both targeting specificity and scaffolding/binding ability for the CRISPR nuclease of the present invention.
  • sgRNAs of the present invention do not exist in nature, i.e., is a non-naturally occurring nucleic acid(s).
  • a "target region”, “target sequence” or “protospacer” in the context of sgRNAs and CRISPR system of the present invention are used herein interchangeably and refers to the region of the target gene, which is targeted by the CRISPR/nuclease-based system, without the PAM. It refers to the sequence corresponding to the nucleotides that precede the PAM (i.e., in 5' or 3' of the PAM, depending of the CRISPR nuclease) in the genomic DNA. It is the sequence that is included into a sgRNA expression construct (e.g., vector/plasmid/AVV).
  • a sgRNA expression construct e.g., vector/plasmid/AVV
  • the CRISPR/nuclease- based system may include at least one (i.e., one or more) sgRNAs, wherein each sgRNA target different DNA sequences on the target gene.
  • the target DNA sequences may be overlapping.
  • the target sequence or protospacer is followed or preceded by a PAM sequence at an (3' or 5' depending on the CRISPR nuclease used) end of the protospacer.
  • the target sequence is immediately adjacent (i.e., is contiguous) to the PAM sequence (it is located on the 5' end of the PAM for SpCas9-like nuclease and at the 3' end for Cpfl-like nuclease).
  • Table 3 Exemplary sgRNA target sites and their adjacent PAM in ATP1A1 exon 4 and surrounding introns.
  • the position of PAM is provided with reference to SEQ ID NO: 2.
  • Identification of SpCas9 PAM sites was performed using CCTopTM (Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J. and Mateo, J.L. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLOS ONE (2015). doi: 10.1371 /journal.pone.0124633) Cpf1 PAM were searched manually. Further non-limiting examples of SpCas9 PAM for additional exons are provided in FIGs 28A-I.
  • G2 [GIATCCAAGCTGCTACAGAAG SEQ ID NO: 28 AGG cs (285-287) (+)
  • sgRNAs target sequences for Cpf1
  • the sgRNA of the present invention comprises a "sgRNA guide sequence" or has a "sgRNA target sequence” which corresponds to the target sequence on the ATP1A1 gene or target polynucleotide sequence that is followed or preceded by a PAM sequence (is adjacent to a PAM).
  • the sgRNA may comprise a "G" at the 5' end of its polynucleotide sequence. The presence of a "G” in 5' is preferred when the sgRNA is expressed under the control of the U6 promoter (Taeyoung KooJungjoon Lee and Jin-Soo Kim Mol Cells. 2015 Jun 30; 38(6): 475-481).
  • the CRISPR/nuclease system of the present invention may use sgRNAs of varying lengths.
  • the sgRNA may comprise a sgRNA guide sequence of at least at least a 10, at least 12 nts, at least a 13 nts, at least a 14 nts, at least a 15 nts, at least a 16 nts, at least a 17 nts, at least a 18 nts, at least a 19 nts, at least a 20 nts, at least a 21 nts, at least a 22 nts, at least a 23 nts, at least a 24 nts, at least a 25 nts, at least a 30 nts, or at least a 35 nts of a target sequence on the ATP1A1 gene or target polynucleotide (such target sequence is followed or preceded by a PAM on the ATP1A
  • the length of the sgRNA is selected based on the specific CRISPR nuclease used.
  • the "sgRNA guide sequence" or “sgRNA target sequence” may be least 17 nucleotides (17, 18, 19, 20, 21 , 22, 23) long, preferably between 17 and 30 nts long, more preferably between 17-22 nucleotides long.
  • the sgRNA guide sequence is between 10- 40, 10-30, 12-30, 15-30, 18-30, or 10-22 nucleotides long.
  • the PAM sequence is "NGG", where "N” can be any nucleotide.
  • the PAM sequence is "TTTN", where "N” can be any nucleotide.
  • sgRNAs may target any region of a target gene (e.g., ATP1A1) which is immediately adjacent (contiguous, adjoining, in 5' or 3') to a PAM (e.g., NGG/TTTN or CCN/NAAA for a PAM that would be located on the opposite strand) sequence.
  • a target gene e.g., ATP1A1
  • PAM e.g., NGG/TTTN or CCN/NAAA for a PAM that would be located on the opposite strand
  • the sgRNA of the present invention has a target sequence that is located in an exon (the sgRNA guide sequence consists of the RNA sequence of the target (DNA) sequence which is located in an exon).
  • the target sequence of the sgRNA is in exon 4 of the ATP1A1 gene.
  • the sgRNA of the present invention has a target sequence that is located in an intron (the sgRNA guide sequence consists of the RNA sequence of the target (DNA) sequence which is located in an intron).
  • the target sequence of the sgRNA is in intron 3 or intron 4 of the ATP1A1 gene.
  • the sgRNA may target any region (sequence) which is followed (or preceded, depending on the CRISPR nuclease used) by a PAM in the ATP1A1 gene or target polynucleotide and which may be used to confer ouabain resistance.
  • a mismatch between a sgRNA guide sequence and target sequence on the gene sequence of interest is also permitted as along as it still allows hybridization of the sgRNA with the complementary strand of the sgRNA target polynucleotide sequence on the targeted gene.
  • a seed sequence of between 8-12 consecutive nucleotides in the sgRNA, which perfectly matches a corresponding portion of the sgRNA target sequence is preferred for proper recognition of the target sequence.
  • the remainder of the guide sequence may comprise one or more mismatches.
  • sgRNA activity is inversely correlated with the number of mismatches.
  • the sgRNA of the present invention comprises 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, more preferably 2 mismatches, or less, and even more preferably no mismatch, with the corresponding sgRNA target gene sequence (less the PAM).
  • the sgRNA nucleic acid sequence is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identical to the sgRNA target polynucleotide sequence in the gene of interest (e.g., ATP1A1).
  • the smaller the number of nucleotides in the sgRNA guide sequence the smaller the number of mismatches tolerated.
  • the binding affinity is thought to depend on the sum of matching sgRNA-DNA combinations.
  • Non-limiting examples of sgRNA target sequences are presented in Table 3 above. Any sgRNA guide sequence can be selected in the target gene, as long as it allows introducing at the proper location, the desired modification(s) (e.g., spontaneous insertions/deletions or selected target modification(s) using one or more patch/donor sequence(s)). Accordingly, the sgRNA guide sequence or target sequence of the present invention may be in coding or non-coding regions of the ATP1A1 gene (i.e., introns or exons). In embodiments, the sgRNA target sequence is selected in intron 3, exon 4 and/or intron 4 of the ATP1A1 gene.
  • the sgRNA guide sequence in combination with the donor/patch sequence allows to replace at least a portion of an in intron and an exon of the endogenous ATP1A1 polynucleotide gene sequence within a cell so as to confer ouabain resistance.
  • SEQ ID NO: 2 presents a fragment of the ATP1A1 polynucleotide gene sequence comprising part of intron 3, exon 4 and part of intron 3 from which sgRNA guide sequence may easily be selected in accordance with the present invention.
  • the complementary strand of the sequence may alternatively and equally be used to identify proper PAM and sgRNA target/guide sequences.
  • the sgRNA of the present invention comprise a guide sequence having a target sequence/guide sequence as set forth in Table 3, FIG. 28 or any one of SEQ ID NOs: 27-30, 37-41.
  • the number of sgRNAs administered to or expressed in a target cell in accordance with the methods of the present invention may be at least 1 sgRNA, at least 2 sgRNAs, at least 3 sgRNAs at least 4 sgRNAs, at least 5 sgRNAs, at least 6 sgRNAs, at least 7 sgRNAs, at least 8 sgRNAs, at least 9 sgRNAs, at least 10 sgRNAs, at least 11 sgRNAs, at least 12 sgRNAs, at least 13 sgRNAs, at least 14 sgRNAs, at least 15 sgRNAs, at least 16 sgRNAs, at least 17 sgRNAs, or at least 18 sgRNAs.
  • the number of sgRNAs administered to or expressed in a cell may be between at least 1 sgRNA and 15 sgRNAs, 1 sgRNA and least 10 sgRNAs, 1 sgRNA and 8 sgRNAs, 1 sgRNA and 6 sgRNAs, 1 sgRNA and 4 sgRNAs, 1 sgRNA and sgRNAs, 2 sgRNA and 5 sgRNAs, or 2 sgRNAs and 3 sgRNAs.
  • a sgRNA having a sgRNA target sequence/guide sequence as shown in Table 3 (SEQ ID NOs: 27-30 and 37-41) or FIG. 28 is used to modify the ATP1A1 gene and confer ouabain resistance in one or more target cells.
  • dCas9-FoKI dimeric nucleases that can recognize extended sequences and edit endogenous genes with high efficiency in human cells.
  • These nucleases comprise a dimerization-dependent wild type Fokl nuclease domain fused to a catalytically inactive Cas9 (dCas9) protein.
  • Dimers of the fusion proteins mediate sequence specific DNA cleavage when bound to target sites composed of two half-sites (each bound to a dCas9 (i.e., a Cas9 nuclease devoid of nuclease activity) monomer domain) with a spacer sequence between them.
  • the dCas9-FoKI dimeric nucleases require dimerization for efficient genome editing activity and thus, use two sgRNAs for introducing a cut into DNA.
  • the recombinant CRISPR nuclease that may be used in accordance with the present invention is i) derived from a naturally occurring Cas; and ii) has a nuclease (or nickase) activity to introduce a DSB (or two SSBs in the case of a nickase) in cellular DNA when in the presence of appropriate sgRNA(s).
  • CRISPR nuclease refers to a recombinant protein which is derived from a naturally occurring Cas nuclease which has nuclease or nickase activity and which functions with the sgRNAs of the present invention to introduce DSBs (or one or two SSBs) in the targets of interest, e.g., the ATP1A1 gene and/or target polynucleotide.
  • the CRISPR nuclease is SpCas9.
  • the CRISPR nuclease is Cpfl
  • the CRISPR nuclease is a Cas9 protein having a nickase activity.
  • Cas9 nickase refers to a recombinant protein which is derived from a naturally occurring Cas9 and which has one of the two nuclease domains inactivated such that it introduces single stranded breaks (SSB) into the DNA. It can be either the RuvC or HNH domain.
  • the Cas protein is a dCas9 protein fused with a dimerization-dependant Fokl nuclease domain.
  • CRISPR nucleases that may be used in accordance with the present invention are provided in Table 2 above.
  • CRISPR nucleases such as Cas9/nucleases cut 3-4bp upstream of the PAM sequence.
  • CRISPR nucleases such as Cpf1 on the other hand, generate a 5' overhang. The cut occurs 19 bp after the PAM on the targeted (+) strand and 23 bp on the opposite strand (Zetsche et al., 2015, PMID 26422227).
  • sgRNA concentration of nuclease and guide RNA
  • CRISPR nuclease proteins are (or are derived from) proteins normally expressed in bacteria, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing CRISPR nuclease recombinant proteins. This has already been done for the embodiments of the present invention described in Examples 1-6. Similarly, donor or patch nucleic acids of the present invention used to introduce specific modifications in ATP1A or in the target polynucleotide may use codon degeneracy (e.g., to introduce new restriction sites for enabling easier detection of the targeted modification).
  • codon chart (Table 4) may be used, in a site-directed mutagenic scheme, to produce nucleic acids encoding the same or slightly different amino acid sequences of a given nucleic acid:
  • Table 5 Exemplary known amino acid modifications in ATP1A1 conferring ouabain resistance (Adapted from Croyle ML, Woo AL, Lingrel JB. Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity-implications for ouabain binding. Eur J Biochem. 1997; 248(2):488-95. PubMed PMID: 9346307).
  • methods of the present invention comprise introducing one or more modification(s) in the ATP1A1 gene by NHEJ or HDR to confer or induce ouabain resistance.
  • ouabain resistance is conferred by introducing a DSB in ATP1A1 gene and by providing an ATP1A1 donor nucleic acid comprising (i.e., encoding) the desired amino acids modification(s).
  • any modification or combination of modifications known to confer ouabain resistance may be introduced in the ATP1A gene (i.e., any nucleic acid encoding an ATP1A1 polypeptide conferring ouabain resistance may be used to replace the corresponding endogenous ATP1A1 polypeptide).
  • the ATP1A1 donor nucleic acids confer ouabain resistance by replacing at least one of amino acid Q118 and N129 in the ATP1A1 gene by a charged amino acid.
  • the charged amino acid is an arginine (R) or an aspartic acid (D).
  • both of Q118 and N129 are replaced by an arginine (R) or an aspartic acid (D).
  • Q118 is replaced by an aspartic acid and N129 is replaced by an arginine.
  • Q118 is replaced by an arginine and N129 is replaced by an aspartic acid.
  • Exemplary sgRNA binding sites surrounding all transmembrane and extracellular regions of ATP1A1 are provided in Table 3 above and FIG. 28.
  • the present invention further provides a method of inducing ouabain resistance in one or more target cells by introducing a DSB in an exon of the ATP1 A1 gene of one or more target cells.
  • such method comprises providing (e.g., transfecting or introducing) the one or more target cells with a sgRNA guide sequence having a target sequence in an exon of the ATP1 A1 gene and a CRISPR-nuclease.
  • the sgRNA target sequence is located in an exon encoding the H1 , H2; H3, H4, H5, H6, H7 H8 or H10 ATP1A1 polypeptide region.
  • the target sequence is located in exon 4.
  • the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acid corresponding to an alanine at position 119 (Aug) in the wild-type ATP1A1 polypeptide (SEQ ID NO: 3). In embodiments, the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acid corresponding to an alanine at position 120 (A120) in the wild-type ATP1A1 polypeptide. In embodiments, the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acid corresponding to a threonine at position 121 (T121) in the wild-type ATP1A1 polypeptide.
  • the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acids corresponding to a glutamic acid at positions 122 (E122), 123 (E123) and/or 124 (E124) in the wild-type ATP1A1 polypeptide.
  • the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acid corresponding to a proline at position 125 (P125) in the wild-type ATP1A1 polypeptide.
  • the ouabain resistant ATP1A1 polypeptide comprises a deletion of the amino acid corresponding to a glutamine at position 126 (Q126) in the wild-type ATP1A1 polypeptide.
  • the ouabain resistant polypeptide comprises a lysine instead of a threonine at amino acid position 121 (T121 K mutation). In embodiments, the ouabain resistant polypeptide comprises an alanine instead of a proline at amino acid position 125 (P125A mutation). In embodiments, the ouabain resistant ATP 1A1 polypeptide comprises a deletion of at least one, at least two, at, least 3, at least 4, at east 5, at least 6, at least 7 or at least 8 amino acids in amino acids corresponding to positions 119 (Aug) and 126 (Q126) in the wild-type ATP1A1 polypeptide. In embodiments, the ouabain resistant ATP1A1 polypeptide comprises a polypeptide sequence as set forth in SEQ ID NO: 5.
  • the present invention further provides nucleic acids encoding sgRNAs, CRISPR nucleases and ouabain resistant ATP1A1 polypeptides of the present invention.
  • RNA expression vectors G1-G4 (500 ng) indicated in Table 7 were transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus. Cells were treated with 0.5 ⁇ ouabain for 7 days starting 3 days post-transfection and monitored for survival and growth. An expression vector encoding EGFP (500ng) was used as a negative control.
  • the G3 sgRNA expression vector 500 ng was co-transfected into K562 cells stably expressing wild-type SpCas9 from the AAVS1 safe harbor locus along with the indicated ssODNs. Cells were treated or not with increasing doses of ouabain for 7 days starting 3 days post-transfection and cell survival and growth was monitored.
  • the HIST1 H2BK locus was targeted to create a C-terminal fusion of H2B with monomeric Azami-Green (mAG).
  • mAG monomeric Azami-Green
  • ouabain treatment resulted in a ⁇ 26-fold increase of targeted cells (FIG. 21).
  • the left homology arm of the donor contains a truncated and non-functional H2B ORF, specific targeting through HDR must have occurred to generate the labeling observed in mAG+ cells (FIG. 21 B).
  • the sequence for the green fluorescent protein Clover after the second codon of the LMNA gene, which encodes the lamin A and lamin C isoforms (22).
  • a versatile and robust approach to increase specificity in a CRISPR nuclease-based system is to use nucleases with inherent or enhanced specificity of action.
  • the Cpfl and high-fidelity Cas9 variants provide an alternative to wild-type SpCas9 and have been shown to be highly specific for their target site based on genome- wide specificity assays in human cells (37-39 and 48-49).
  • Purified human CD34+ cells were electroporated with preformed SpCas9 ribonucleoprotein complexes (RNPs) containing synthetic crRNAs and tracrRNAs targeting ATP1A1 and expanded ex vivo in presence or absence of ouabain.
  • RNPs SpCas9 ribonucleoprotein complexes
  • TIDE analysis revealed the presence of in-frame deletions in up to 50% of alleles in presence of ouabain in comparison to -10% in untreated cells (FIG. 26).
  • cells edited via HDR at the ATP1 A1 locus were efficiently enriched using ouabain (FIG. 26).
  • K562 and U20S cells were obtained from the ATCC and maintained at 37 °C under 5% CO2 in RPMI medium supplemented with 10% FBS, penicillin-streptomycin and GlutaMAX. Cells were transfected using the Amaxa 4D-NucleofectorTM (Lonza) per manufacturer's recommendations. Ouabain (Sigma) was added directly to the culture medium.
  • Genomic DNA from 2.5E5 cells was extracted with 250 ⁇ of QuickExtractTM DNA extraction solution (Epicentre) per manufacturer's recommendations.
  • the ATP1A1 locus was amplified by 30 cycles of PCR using the following primers: 5'- CACTTGTAAGAGCATCTACAACG-3' (SEQ ID NO: 449) and 5'-GGATTAACATCTGCTCGTGCAGC-3' (SEQ ID NO: 450).
  • the AAVS1 locus was amplified by 30 cycles of PCR using the following primers: 5'- CCCCTTACCTCTCTAGTCTGTGC-3' (SEQ ID NO: 451) and 5'- CTCAGGTTCTGGGAGAGGGTAG-3' (SEQ ID NO: 452).
  • the HPRT1 locus was amplified by 30 cycles of PCR using the following primers: 5'- GGTGTGGAAGTTTAATGACTAAGAGG-3' (SEQ ID NO: 453) and 5'-TCACTGTAACCAAGTGAA ATGAAAGC-3' (SEQ ID NO: 454).
  • Assays were performed with the SurveyorTM mutation detection kit (Transgenomics) according to the manufacturer's protocol. Samples were separated on 10% PAGE gels in TBE buffer.
  • PCR products were purified and digested with the corresponding enzyme and resolved by 10% PAGE. Gels were imaged using a ChemiDocTM MP (Bio-Rad) system and quantifications were performed using Image lab software (Bio-Rad) as described (13). TIDE analysis was performed as described (35).
  • CRISPR/Cas9 reagents The CAG-driven human codon optimized Cas9 nuclease vectors pCas9_GFP (Addgene #44719) was used in all transient transfection experiments. The expression cassette was transferred to AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene #68375) in order to establish the K562 cell line constitutively expressing Cas9. All sgRNA expression vectors were built in the MLM3636 (Addgene #43860) backbone.
  • Target sequences for ATP1A1-G1 (GAACTCACATTATCGTTTTG, SEQ ID NO: 27), AT1A1-G2 (GATCCAAGCTGCTACAGAAG, SEQ ID NO: 28), ATP1A1-G3 (GAGTTCTGTAATTCAGCATA, SEQ ID NO: 29), ATP1A1-G4 (GTTCCTCTTCTGTAGCAGCT, SEQ ID NO: 30), HPRT1-S1R (FIGs. 19-20) (GATGTGATGAAGGAGATGGG, SEQ ID NO: 455), HPRT1-S2L (FIG.
  • sgRNA sequences are provided as follows: ATP1A1-G1 (SEQ ID NO: 23); AT1A1-G2 (SEQ ID NO: 24); ATP1A1-G3 (SEQ ID NO: 25); ATP1A1-G4 (SEQ ID NO: 26); HPRT1-S1R (SEQ ID NO: 465); HPRT1-S2L (SEQ ID NO: 466) and HIST1H2BK (SEQ ID NO: 467).
  • RD (CAATGTTACTGTGGATTGGAGCGATTCTTTGTTTCTTGGCTTATAGCATCAGAGCTGCTACAGAA GAGGAACCTCAAAACGATGACGTGAGTTCTGTAATTCAGCATATCGATTTGTAGTACACATCAGATATCTT, SEQ ID NO: 44)
  • CRISPR/Cpf1 reagents The human codon optimized Cpf1 ORF from the nuclease vector pY010, a gift from Feng Zhang (Addgene plasmid # 69982) (41), was transferred to AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid # 68375) in order to establish the K562 cell line constitutively expressing Cpf1 from a CAG promoter.
  • a crRNA expression vector was built in the pUC19 backbone by linking the AsCpfl 5'DR to a human U6 promoter (41). The guide sequences where cloned downstream of the 5'DR.
  • Target sequences for ATP1A1-G5 (TTTCTTGGCTTATAGCATCC, SEQ ID NO: 37), ATP1A1-G6 (TTGGCTTATAGCATCCAAGC, SEQ ID NO: 38), ATP1A1-G7 (AGGTTCCTCTTCTGTAGCAG, SEQ ID NO: 39), ATP1A1-G8 (GAGGTTCCTCTTCTGTAGCA, SEQ ID NO: 40), and ATP1A1-G9 (TAGTACACATCAGATATCTT, SEQ ID NO: 41 were chosen manually by scanning the target regions for TTTN PAMs. Desalted ssODN were synthesized as ultramers (IDT) at 4 nmole scale
  • All plasmid donor sequences contain short homology arms ( ⁇ 1 kb) and have been modified in order to prevent their cleavage by Cas9 as described (9).
  • CD34 + cells Targeting in primary human cord blood (CB) CD34 + cells.
  • CB samples from volunteer donors were acquired after written informed consent according to the guidelines approved by Hema-Quebec and Laval University.
  • Mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare) density centrifugation.
  • CD34 + haematopoietic stem and progenitor cells HSPCs
  • HSPCs haematopoietic stem and progenitor cells
  • Purity of CD34 + cells was assessed and cells were cryopreserved in Cryostor CS10 (StemCell Technologies).
  • CD34 + HSPCs were cultured in StemSpan ACF (StemCell Technologies) supplemented with 100ng/ml SCF, 100ng/ml FLT3-L, 50ng/ml TPO, 10pg/ml LDL, 35nM UM171. The medium was changed every 3-4 days. Ouabain (0.5 ⁇ ) was added directly to the culture media change at day 6 post-transfection. For enrichment via HDR, ouabain was kept in the culture during the entire selection period. For enrichment via NHEJ, ouabain was added for the first week. Cultured CD34 + cells (100 000- 200 000 per sample) were spun at 300g for 10 minutes, washed in PBS and resuspended in P3 Nucleofector solution (Lonza).
  • crRNA and tracrRNA were resuspended to 200 ⁇ stock solutions in Nuclease-Free IDTE Buffer.
  • the two RNA oligos were mixed in equimolar concentrations, heated at 95°C for 5 minutes and allowed to cool to RT on bench top.
  • sgRNA G3 was transcribed in vitro using the EnGenTM sgRNA Synthesis kit (NEB) as per the manufacturer's instructions using the following oligo (5 ' - TTCTAATACGACTCACTATAGAGTTCTGTAATTCAGCATAGTTTTAGAGCTAGA-3 ' ; SEQ ID NO: 458).
  • In vitro transcription products were purified using RNA Clean & Concentrator-25 (Zymo Research) and eluted in nuclease- free water.
  • a genomic safe harbor is a chromosomal site where transgenes can be stably and reliably expressed in the tissue of interest without adversely affecting endogenous gene structure or expression (55).
  • An ideal site for transgene insertion should allow (i) robust and stable transgene expression across different cell types; (ii) no transcriptional perturbation owing to the transgene cassette and (iii) no disruption of essential regulatory or coding sequences due to the transgene cassette, and (iv) no physiological impact on the organism (56,57).
  • the standard locus for transgenesis via gene targeting in mouse embryonic stem cells, rosa26, is a prototypical GSH (55).
  • AAVS1 In human cells, the site known as AAVS1, found within the first intron of the PPP1R12C gene is considered a "safe harbor" locus (56, 54, see also DeKelver et al. US 8,110,379 B2).
  • the efficacy of targeted integration at this locus is subject to the same limitations imposed by the cell type and cell-cycle stage as these factors play a major role in determining the fate of genome editing.
  • one requires the use of classical selection markers.
  • ATP1A1 as a "genomic safe harbor” (see Example 9) can be coupled to the co-selection process in order to increase the levels of targeted integration at a distinct locus of interest.
  • mScarlet red fluorescent protein
  • mNeonGreen antiparallel v1 donor see Example 9
  • ouabain >67% of the cells were double positive for both FPs indicating that selection for targeted integration at ATP1A1 led to the co-selection for targeted integration at LMNA (FIG. 36).
  • co-selection process can be initiated through NHEJ or HDR processes independently.
  • the co-selection strategy markedly enriches for cells modified via homologous recombination, a clear limitation in the field.
  • Methods of the present invention should be compatible with any engineered nuclease platforms and any cell type when using a ubiquitously expressed gene such as ATP1A1. Perhaps more importantly, robust selection is achieved without the use of exogenous markers making the process compatible with therapeutic applications.
  • transporting subunit alpha 1 isoform c This variant represents the longest transcript and encodes the longest isoform.
  • SEQ ID NO: 2 nts Part of genomic polynucleotide sequence of human ATP1A1 , from Gene ID 476. 1- nts 1-100 : intron 3; 101-304: exon 4; and 305-404 intron 4
  • SEQ ID NO: 6 aa Ouabain resistant human ATP1 A1 consensus sequence including all amino acids which can be mutated to confer ouabain resistance (includes WT aa and mutant aa in Table 4 which lists known amino acid mutations in ATP1 A1 conferring ouabain resistance. Includes aa in novel variants identified herein).
  • SEQ ID NO: 23 nts Single guide RNA (sgRNA) 1 (ATP1A1-G1) sequence including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)
  • SEQ ID NO: 24 nts Single guide RNA (sgRNA) 2 (ATP1A1-G2) including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)
  • SEQ ID NO: 25 nts Single guide RNA (sgRNA) 3 (ATP1A1-G3) including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)
  • SEQ ID NO: 26 nts Single guide RNA (sgRNA) 4 (ATP1A1-G4) including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)
  • SEQ ID NO: 27 nts target sequence (sgRNA) 1 (ATP1A1-G1)
  • SEQ ID NO: 28 nts target sequence (sgRNA) 2 (ATP1A1-G2) ATP1A1 (lacking added G in 5' for U6 promoter)
  • SEQ ID NO: 29 nts target sequence (sgRNA) 3 (ATP1A1-G3)
  • SEQ ID NO: 30 nts target sequence (sgRNA) 4 (ATP1A1-G4)
  • SEQ ID NO: 32 nts Single guide RNA (sgRNA) 5 (ATP1A1-G5) including
  • SEQ ID NO: 33 nts Single guide RNA (sgRNA) 6 (ATP1A1-G6) including CRISPR nuclease (AsCpfl) recognition sequence (Single CRISPR guide RNA -5' Direct repeat)
  • SEQ ID NO: 34 nts Single guide RNA (sgRNA) 7 (ATP1A1-G7) including CRISPR nuclease recognition
  • SEQ ID NO: 35 nts Single guide RNA (sgRNA) 8 (ATP1A1-G8) including CRISPR nuclease recognition
  • SEQ ID NO: 36 nts Single guide RNA (sgRNA) 9 (ATP1A1-G9) including CRISPR nuclease (AsCpfl) recognition sequence (Single CRISPR guide RNA -5' Direct repeat)
  • SEQ ID NO: 37 nts target sequence (sgRNA) 5 (ATP1A1-G5) (DNA)- (AsCpfl)
  • SEQ ID NO: 38 nts target sequence (sgRNA) 6 (ATP1A1-G6) (DNA)- (AsCpfl)
  • SEQ ID NO: 39 nts target sequence (sgRNA) 7 (ATP1A1-G7) (DNA)- (AsCpfl)
  • SEQ ID NO: 40 nts target sequence (sgRNA) 8 (ATP1A1-G8) (DNA)- (AsCpfl)
  • SEQ ID NO: 41 nts target sequence (sgRNA) 9 (ATP1A1-G9) (DNA) - (AsCpfl)
  • SEQ ID NO: 42 nts CRISPR recognition sequence for AsCpfl (5' direct-repeat RNA sequence)
  • SEQ ID NO: 46 aa AsCpfl protein sequence (derived from Acidaminococcus species-basic sequence, no histag or NLS)
  • SEQ ID NOs: nts Primers for amplification of (i) ATP1A1 locus (SEQ ID NOs:449-450); (ii) The AAVS1 449-454 locus (SEQ ID NOs: 451-452); (Hi) The HPRT1 locus (SEQ ID NOs: 453-454)
  • SEQ ID NO: 461 nts Human ATP 1 A3 wild-type ORF (cDNA): NMJ52296.4. ATPase Na+/K+
  • SEQ ID NO: 465 nts Single guide RNA (sgRNA) HPRT1-S1R sequence including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)- FIGs. 19 and 20
  • SEQ ID NO: 466 nts Single guide RNA (sgRNA) HPRT1-S2L sequence including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)- FIG. 13
  • SEQ ID NO: 467 nts Single guide RNA (sgRNA) HIST1H2BK sequence including CRISPR nuclease recognition sequence (chimeric guide RNA scaffold)- FIGs. 21 and 29
  • SEQ ID NO: 468 nts AAVS1 (T2) target sequence- FIGs. 12 and 22
  • SEQ ID NO: 469 nts Single guide RNA (sgRNA) AAVS1-T2 sequence including CRISPR nuclease
  • SEQ ID NO: 471 nts Single guide RNA (sgRNA) LMNA-G2 sequence including CRISPR nuclease
  • SEQ ID NO: 472 nts Splicing acceptor-SA-2A-EGFP-pA donor sequence containing homology arms to
  • SEQ ID NO: 476 nts ATP1 A1 donor sequence (TurboGFP, antiparallel orientation, Figures 31 and 32 and
  • SEQ ID NO: 477 nts DNA donor sequence (V1-NeonGreen) containing expression cassettes for green fluorescent protein flanked by homology arms for targeted integration within intron 4 01 ATP1A1- FIG. 33
  • SEQ ID NO: 478 nts DNA donor sequence (V2-NeonGreen) containing expression cassettes for green fluorescent protein flanked by homology arms for targeted integration within intron 4 of ATPVAV-Exemple 9 and FIG. 33
  • SEQ ID NO: 479 nts DNA donor sequence (LMNA-mScarlet)-Example 10 and FIG. 36
  • SEQ ID NO: 480 nts DNA donor sequence for HPRT (PGK-EGFP)- FIG. 23
  • Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR- Cas system. Cell. 2015; 163(3)759-71. doi: 10.1016 j.cell.2015.09.038. PubMed PMID: 26422227; PMCID:
  • Kleinstiver BP Tsai SQ
  • Prew MS Nguyen NT
  • Welch MM Welch MM
  • Lopez JM McCaw ZR
  • Aryee MJ Joung JK.

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Abstract

L'invention concerne des procédés et des produits pour la modification (par exemple la délétion, l'insertion ou la substitution) d'un gène cible dans une cellule. Dans un aspect, le procédé implique l'introduction d'une modification dans un locus d'un gène endogène (par exemple ATP1A), ce qui confère une résistance à un médicament (par exemple l'ouabaïne ou un dérivé correspondant) et la co-introduction d'une modification dans un gène cible d'intérêt. Une sélection basée sur un médicament peut être utilisée pour enrichir les cellules comprenant lesdits gènes modifiés au sein d'une population de cellules, ce qui permet d'effectuer une sélection pour de tels gènes modifiés. L'invention concerne également de nouveaux polypeptides, y compris de nouveaux polypeptides ATP1A1, qui confèrent une résistance à un médicament (par exemple l'ouabaïne), ainsi que des acides nucléiques correspondants codant pour ceux-ci, ainsi que des vecteurs et des cellules hôtes les comprenant.
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WO2023015376A1 (fr) * 2021-08-11 2023-02-16 UNIVERSITé LAVAL Modification ciblee sur mtor et ses utilisations
WO2023150393A3 (fr) * 2022-02-07 2023-09-21 Ensoma, Inc. Modifications de mgmt résistante aux inhibiteurs et modification d'acides nucléiques codant mgmt

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CN110305873A (zh) * 2019-01-23 2019-10-08 福建上源生物科学技术有限公司 共编辑标记ben-1sgRNA靶位点及其CRISPR/Cas9共编辑系统和应用
WO2020230976A1 (fr) * 2019-05-14 2020-11-19 서울대학교 산학협력단 Procédé de sélection de cellules génétiquement modifiées à partir de cellules souches pluripotentes indifférenciées
WO2023015376A1 (fr) * 2021-08-11 2023-02-16 UNIVERSITé LAVAL Modification ciblee sur mtor et ses utilisations
WO2023150393A3 (fr) * 2022-02-07 2023-09-21 Ensoma, Inc. Modifications de mgmt résistante aux inhibiteurs et modification d'acides nucléiques codant mgmt

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