Methods for increasing the efficiency of homology-directed repair (HDR) in cellular genomes

Abstract

To provide a method for increasing the efficiency of homology-directed repair (HDR) in a cellular genome.SOLUTION: (a) introducing into a cell (i) a nuclease and (ii) a donor nucleic acid comprising a modified sequence to be inserted into the genome, and (b) subjecting the cell to a temperature shift from 37 °C to a lower temperature, wherein the nuclease cleaves the genome at a cleavage site in the cell and the donor nucleic acid directs repair of the genomic sequence with the modified sequence by an increased rate of HDR.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 437,042, filed December 20, 2016, which is incorporated herein in its entirety for all purposes. [Background technology]

[0002] Background of the Invention Various methods for DNA target cleavage of genomic sequences have been described in the art. These target cleavage events can be used to induce targeted mutagenesis, induce targeted deletion of cellular DNA sequences, and promote targeted recombination at a specific chromosomal locus. These methods often involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks in target DNA sequences, so that repair of the cleavage by error-generating processes such as non-homologous end joining (NHEJ) or homology-directed repair (HDR) can result in gene inactivation or insertion of a desired exogenous sequence. Cleavage can be achieved by using specific nucleases, such as engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system, together with engineered single guide RNAs (sgRNAs) to guide specific cleavage.

[0003] The efficiency of genome modification at specific target locations by the HDR process is relatively low in cells, and therefore there remains a need for methods to increase the efficiency of homology-directed repair (HDR) in cellular genomes. Summary of the Invention

[0004] SUMMARY OF THE INVENTION In one embodiment, the present invention provides a method for increasing the efficiency of homology-directed repair (HDR) in the genome of a cell, comprising: (a) introducing into the cell (i) nuclease and (ii) donor nucleic acid comprising the modified sequence to be inserted into the genome; and (b) subjecting the cell to a temperature shift from 37 ° C to a lower temperature, wherein the nuclease cuts the genome at the cut site in the cell, and the donor nucleic acid guides the repair of the genome sequence by the modified sequence through the increased HDR rate.For example, the homology-directed repair (HDR) rate is increased by at least 1.5 times.In some cases, the homology-directed repair (HDR) rate is increased by at least 2 times.

[0005] In some aspects, the lower temperature is between 28°C and 32°C. Optionally, the lower temperature is between 30°C and 33°C. For example, the cells are grown at the lower temperature for at least 24 hours, or at least 48 hours, e.g., 1 to 5 days (1 day, 2 days, 3 days, 4 days, or 5 days). Optionally, the cells are grown at 37°C after the temperature shift.

[0006] In some aspects, the cell is a eukaryotic cell, such as a mammalian cell. In certain embodiments, the cell is a stem cell, such as an induced pluripotent stem cell (iPSC). In another specific embodiment, the cell is a primary cell.

[0007] In some aspects, the nuclease used in the present invention includes all DNA sequence-specific endonucleases or RNA-guided DNA endonucleases. Optionally, the nuclease is a CRISPR nuclease selected from Cas nuclease or Cpf1 nuclease. For example, the nuclease is a Cas9 nuclease. For example, the CRISPR nuclease (e.g., Cas9) is introduced into cells in DNA format (e.g., DNA encoding Cas9 nuclease and sgRNA) or in RNA format (e.g., sgRNA / Cas9RNP or sgRNA / Cas9mRNA) together with sgRNA. Optionally, the sgRNA is synthetic and chemically modified. In some aspects, the donor nucleic acid contains symmetric homology arms. Optionally, the donor nucleic acid is complementary to the DNA strand in the genome that is cut by the nuclease.

[0008] In one aspect, the nuclease used in the present invention is a zinc finger nuclease (ZFN). In another aspect, the nuclease used in the present invention is a TALE nuclease (TALEN).

[0009] In certain embodiments, the present invention provides isolated cells produced by the above methods.

[0010] In certain embodiments, the present invention provides a pharmaceutical composition comprising the isolated cells produced by the above-described methods.

[0011] In one embodiment, the present invention provides a method for providing a protein of interest to a subject in need thereof, the method comprising: (a) introducing a donor nucleic acid encoding the protein of interest into a cell according to the methods described above; and (b) introducing the cell into the subject to express the protein of interest in the subject.

[0012] In one embodiment, the present invention provides a method for increasing the efficiency of homology-directed repair (HDR) in a cell's genome, comprising: introducing into the cell (i) a nuclease; and (ii) a donor nucleic acid comprising a symmetric homology arm, complementary to the DNA strand in the genome cleaved by the nuclease, and comprising a modified sequence to be inserted into the genome at a distance of more than 10 base pairs from the cleavage site; wherein the nuclease cleaves the genome at the cleavage site in the cell, and the donor nucleic acid guides the repair of the genome sequence with the modified sequence by an increased HDR rate. For example, the homology-directed repair (HDR) rate is increased by at least 1.5 times, or at least 2 times. Optionally, the method further comprises subjecting the cell to a temperature shift from 37 ° C to a lower temperature (for example, between 28 ° C and 35 ° C, or between 30 ° C and 33 ° C). For example, the cells are grown at the lower temperature for at least 24 hours, or at least 48 hours, for example, 1 to 5 days (1 day, 2 days, 3 days, 4 days, or 5 days). Optionally, the cells are grown at 37°C after the temperature shift. In one aspect, the cells are eukaryotic cells, such as mammalian cells. In a specific embodiment, the cells are stem cells, such as induced pluripotent stem cells (iPSCs). In another specific embodiment, the cells are primary cells. In one aspect, the nuclease used in the present invention is a CRISPR nuclease selected from Cas nuclease or Cpf1 nuclease. For example, the nuclease is Cas9 nuclease. For example, CRISPR nuclease (e.g., Cas9) is introduced into cells in DNA format (e.g., DNA encoding Cas9 nuclease and sgRNA) or in RNA format (e.g., sgRNA / Cas9RNP or sgRNA / Cas9mRNA) together with sgRNA. In some aspects, the nuclease used in the present invention is a zinc finger nuclease (ZFN). In other aspects, the nuclease used in the present invention is a TALE nuclease (TALEN). [Brief explanation of the drawings]

[0013] [Figure 1]Figures 1a-b show the single-stranded oligonucleotide (ssODN) donor design, droplet digital PCR probe, and primer design for gene editing and mutation detection at the CAMK2D locus. (a) Two guide RNAs (CAMK-CR1 and CAMK-CR2) were designed to specifically target CAMK2D exon 2. CAMK-CR1 and CAMK-CR2 overlap by 14 nucleotides and cleave DNA to introduce the same sequence alteration via HDR. (b) Two ssODN HDR donors (C-CR2 and C-CR2-Asym) were designed to introduce a kinase knockout K43R mutation (AAA to AGG) and four silent mutations into exon 2 of the CAMK2D locus. The ssODN donor C-CR2 is the (+) strand HDR donor and has balanced homology arms on either side of the target mutation (5'-73nt and 3'-72nt, respectively). C-CR2-Asym is the negative strand HDR donor, with homology arms of different lengths (5'-93nt and 3'-36nt, respectively) complementary to the non-target strand of the guide RNA. To prevent subsequent re-cleavage, both donor oligos C-CR2 and C-CR2-Asym introduce three silent mutations within the guide CAMK-CR1 recognition site and one silent mutation within the PAM site. C-CR2 and C-CR2-Asym introduce four silent mutations within the guide CAMK-CR2 recognition site. A pair of primers and allele-specific probes conjugated with Vic or Fam fluorophores were also designed to detect unaltered wild-type alleles and mutant sequence conversion events, respectively. To ensure amplification of the appropriate locus, the forward primer was designed to anneal within the donor sequence, while the reverse primer was designed to anneal outside the donor sequence.

[0014] [Figure 2]Figures 2a-c show an optimized method for co-delivery of a single-stranded oligonucleotide donor (ssODN) and sgRNA / Cas9 mRNA to perform HDR at the CAMK2D locus in mc-iPSCs. sgRNA CAMK-CR1 or CAMK-CR2, Cas9 mRNA, and ssODN donor C-CR2 were co-transfected into mc-iPSCs using EditPro® RNA transfection reagent. (a) The percentage of wild-type and mutant alleles from transfected cells was detected by droplet digital PCR (ddPCR) using a wild-type allele-specific fluorescent probe (VIC) and a mutant allele-specific fluorescent probe (FAM). The fluorescence intensity of each droplet in the sample is plotted against the number of droplets. Droplets with fluorescence intensity above the pink threshold line are counted as positive for the target allele. The bottom panel (green) represents droplets with the wild-type allele, and the top panel (blue) represents alleles for which HDR occurred. Data shown are from one representative experiment using two sgRNAs at the best concentration of 10 pmol of ssODN. (b) Quantification of mutant allele frequency. Data are presented as the mean ± SEM from four independent experiments. sgRNA CAMK-CR2 consistently resulted in HDR in more than 15% of all alleles. (c) Quantification of the desired nucleotide changes by next-generation sequencing using the same gRNA and donor used in the ddPCR experiments. Each bar represents one of six nucleotide changes contained in the donor oligo. The data show that complete sequence conversion occurred across the targeted region and at a frequency consistent with the ddPCR results. Two A-to-G changes not directly measured by ddPCR were also incorporated, albeit at a lower frequency compared to the A-to-G changes closer to the CRISPR cut site. Data shown are the average percent of the desired base changes at each precise genomic coordinate from four independent experiments.

[0015] [Figure 3]Figures 3a-b show the effects of "cold shock" and ssODN HDR donor design on HDR efficiency at the CAMK2D locus in mc-iPSCs, as determined by NGS. To achieve HDR at the CAMK2D locus, varying amounts of ssODN C-CR2 or C-CR2-Asym were delivered into mc-iPSCs along with Cas9 mRNA and sgRNA CAMK-CR1 or CAMK-CR2. Experiments were performed at various temperatures at 24-h intervals, as described in "Materials and Methods": PL1: 37°C-37°C, PL2: 37°C-32°C-37°C, PL3: 37°C-32°C-32°C. (a) For each treatment (CAMK-CR1 with C-TR2 or C-TR2-Asym, CAMK-CR2 with C-TR2 or C-TR2-Asym), HDR events using 10 pmol of ssODN HDR donor were determined by NGS as described in Materials and Methods. Data presented are the average percentage of HDR events (C-CR2: 8 replicates from 3 independent experiments; C-CR2-Asym: 6 replicates from 2 independent experiments). HDR types were categorized into three groups based on the resulting sequence surrounding the region of the mutation of interest: Full HDR: All of the desired base changes are present without any re-edited indels; Edited HDR: One or more desired base changes are present, along with the presence of re-edited indels; Partial HDR: Some, but not all, of the desired base changes are present, without any indels. The data demonstrate that increased HDR can be achieved by "cold-shocking" cells, and that the majority of the increase is in the "full HDR" category. The significance of differences in overall HDR efficiency among the three temperature conditions for each gRNA and ssODN treatment was analyzed by one-way ANOVA (one-way ANOVA P<0.0001 for all gRNA and ssODN treatments, P values ​​for follow-up Dunnett's multiple comparisons are shown in the figures). HDR from the 30 pmol ssODN and no oligo treatments is shown in Table 4.(b) Full HDR events for each treatment (CAMK-CR1 with C-TR2 or C-TR2-Asym, CAMK-CR2 with C-TR2 or C-TR2-Asym) were plotted to compare the full HDR frequency between the two ssODN designs. Data presented are the mean percentage of full HDR events ± SEM (six biological replicates from two independent experiments). Differences in full HDR frequency between the two ssODN designs in each treatment group were assessed by Student's T-test, and P values ​​are shown in the figure. Across all temperature conditions, the (+) strand ssODN C-CR2 promoted more full HDR than the (-) strand ssODN C-CR2-Asym.

[0016] [Figure 4]Figures 4a-4c show the guide RNA and single-stranded oligonucleotide (ssODN) donor designs for gene editing at the TGFBR1 locus. (a) Two guide RNAs, TR-CR2 and TR-CR3, were designed to specifically target TGFBR1 Exon 4 and introduce different sequence changes via HDR. TR-CR3 is located 39 nucleotides downstream of TR-CR2. (b) Two ssODN HDR donors (T-CR2 and T-CR2-Asym) were designed to introduce silent mutations 12 bp upstream of the guide RNA TR-CR2 target site and three silent mutations within the guide RNA recognition sequence to prevent re-editing of the HDR-converted sequence. T-CR2 is the (+) strand HDR donor, with balanced homology arms on either side of the desired mutation (5'-73nt and 3'-74nt, respectively) that are complementary to the guide RNA target cleavage strand. T-CR2-Asym has homology arms of different lengths (5'-93nt and 3'-36nt, respectively) and is the negative strand HDR donor complementary to the non-target strand of the guide RNA. (c) Two ssODN donors (T-CR3 and T-CR3-Asym) were designed to contain a known SNP 12 bp upstream of the guide RNA TR-CR3 target site, two silent mutations within the guide RNA recognition sequence, and one silent mutation within the TR-CR3 PAM site to prevent re-editing of the HDR-converted sequence. T-CR3 has balanced homology arms (5'-73nt and 3'-72nt, respectively) on either side of the target mutation, and is the positive strand HDR donor complementary to the guide RNA target strand. T-CR3-Asym has unbalanced homology arms (5'-86nt and 3'-36nt, respectively) on either side of the target mutation and is an HDR donor with a (-) strand complementary to the non-target strand of the guide RNA.

[0017] [Figure 5]Figures 5a-b show the effects of "cold shock" and ssODN HDR donor design on HDR efficiency at the TGFBR1 locus in mc-iPSCs. The ssODN HDR donor and sgRNA were co-delivered with Cas9 mRNA into mc-iPSC cells to achieve HDR at the TGFBR1 locus. Experiments were performed at various temperatures at 24-hour intervals, as described in "Materials and Methods": PL1: 37°C-37°C-37°C, PL2: 37°C-32°C-37°C, PL3: 37°C-32°C-32°C, PL4: 32°C-32°C-32°C. (a) For each treatment (TR-CR2 with T-TR2 or T-TR2-Asym, TR-CR3 with T-TR3 or T-TR3-Asym), HDR events using 10 pmol of ssODN HDR donor were determined by NGS as described in Materials and Methods. Data presented are the average percentage of HDR events (four replicates from three independent experiments). HDR types were categorized into three groups based on the resulting sequence surrounding the region of the mutation of interest: Full HDR: All of the desired base changes are present without any re-edited indels; Edited HDR: One or more desired base changes are present along with the presence of re-edited indels; Partial HDR: Some, but not all, of the desired base changes are present without any indels. The significance of differences in overall HDR efficiency among the three temperature conditions for each gRNA and ssODN treatment was analyzed by one-way ANOVA (one-way ANOVA P > 0.05, follow-up Dunnett's multiple comparison P values ​​are shown in the figure). HDR from 30 pmol ssODN and no oligo treatments is shown in Table 5. (b) Full HDR events using 10 pmol ssODN HDR donor for each treatment (TR-CR2 with T-TR2 or T-TR2-Asym, TR-CR3 with T-TR3 or T-TR3-Asym) were plotted to compare the full HDR frequency between the two ssODN designs. Data presented are the mean percent of full HDR events ± SEM (three independent experiments with four replicates).The difference in the frequency of complete HDR between the two ssODN designs in each treatment group was assessed by Student's T-test, and the P values ​​are shown in the figure. Across all temperature conditions, the (+) ssODN strands T-CR2 and T-CR3 promoted more complete HDR than the (-) ssODN strands T-CR2-Asym and T-CR3-Asym, respectively.

[0018] [Figure 6] Figure 6 shows that "cold shock" enhances HDR efficiency at the CAMK2D locus in HEK293T cells. To achieve HDR at the CAMK2D locus, various amounts of ssODN C-CR2 were delivered into HEK293T cells together with Cas9 mRNA and sgRNA CAMK-CR1 or CAMK-CR2 using the same transfection conditions as mc-iPSCs. Experiments were performed at various temperatures at 24-h intervals as described in "Materials and Methods": PL1: 37°C-37°C-37°C, PL2: 37°C-32°C-37°C, PL3: 37°C-32°C-32°C. (a) HDR events using 10 pmol of ssODN HDR donor for each treatment were determined by NGS as described in "Materials and Methods." Data are presented as the average percentage of HDR events from three replicates. HDR types were classified into three groups based on the resulting sequence surrounding the region of the mutation of interest: Full HDR: All of the desired base changes occurred, with no indels; Editing HDR: One or more of the desired base changes occurred, but with indels; Partial HDR: Some, but not all, of the desired base changes occurred, with no indels. The significance of differences in overall HDR efficiency between the three temperature conditions for each gRNA and ssODN treatment was analyzed by one-way ANOVA. (One-way ANOVA: CAMK-CR1 with C-CR2, P = 0.0041; CAMK-CR2 with C-CR2, P = 0.0469; P values ​​for follow-up Dunnett's multiple comparisons are shown in the figure.) HDRs from the 30 pmol ssODN and no oligo treatments are shown in Table 7.

[0019] [Figure 7] Figures 7a-c show the expression of pluripotency markers in mc-iPSCs after "cold shock." Mc-iPSCs were grown at various temperatures at 24-hour intervals as described in the Supplementary Methods: PL1: 37°C-37°C-37°C, PL2: 37°C-32°C-37°C, PL3: 37°C-32°C-32°C. Cells were then stained with pluripotency-specific antibodies as described in the Supplementary Methods: (a) SSEA3 (green), (b) Nanog (green), and (c) OCT4 (green). Cells were also co-stained with Hoechst to label nuclei (blue).

[0020] [Figure 8]Figure 8 shows that "cold shock" enhances HDR efficiency at the CAMK2D locus in mc-iPSCs, as determined by NGS. Thirty pmol of ssODN C-CR2 was delivered to mc-iPSCs along with Cas9 mRNA and sgRNA CAMK-CR1 or CAMK-CR2 to achieve HDR at the CAMK2D locus. Experiments were performed at various temperatures at 24-hour intervals, as described in the "Materials and Methods" section: PL1: 37°C-37°C-37°C, PL2: 37°C-32°C-37°C, PL3: 37°C-32°C-32°C, PL4: 32°C-30°C-37°C, PL5: 37°C-30°C-30°C, PL6: 37°C-28°C-37°C, PL7: 37°C-28°C-28°C. (a) HDR events for each treatment were determined by NGS, as described in the "Materials and Methods" section. Data presented are the average percentage of HDR events from two replicates. HDR types were categorized into three groups based on the resulting sequences surrounding the region of the mutation of interest: Full HDR: All of the desired base changes are present without any re-edited indels. Edited HDR: One or more of the desired base changes are present, along with the presence of re-edited indels. Partial HDR: Some, but not all, of the desired base changes are present, without any indels. The low percentage of edited HDR and partial HDR sequences in the no-oligo treatment represents the background error rate for next-generation sequencing. No full HDR was detected in the no-oligo treatment. The data demonstrate that increased HDR can be achieved by "cold-shocking" the cells, and that the majority of the increase is in the "full HDR" category. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention In one aspect, the present invention relates to a method for increasing the efficiency of homology-directed repair (HDR) in a cellular genome, for example, by using CRISPR / Cas9 technology. As described in the Examples, the applicants demonstrated that low HDR rates (1-20%) in cells (e.g., PSCs and HEK293 cells) can be increased by 2-10 times by "cold-shocking" the cells at a lower temperature after transfection. The method also increases the proportion of loci that undergo complete sequence conversion across the donor sequence, i.e., "full HDR," as opposed to partial sequence conversion ("partial HDR"), in which nucleotides more distal from the CRISPR cleavage site are less efficiently incorporated. Furthermore, the Examples demonstrate that the structure of the single-stranded DNA oligo donor can significantly affect the fidelity of HDR, and that oligos that are symmetrical about the CRISPR cleavage site and complementary to the target strand are more efficient at inducing "full HDR" compared to asymmetric, non-target strand-complementary oligos.

[0022] In one embodiment, the present invention provides a method for increasing the efficiency of homology-directed repair (HDR) in the genome of a cell, comprising: (a) introducing into the cell (i) nuclease and (ii) donor nucleic acid comprising the modified sequence to be inserted into the genome; and (b) subjecting the cell to a temperature shift from 37 ° C to a lower temperature, wherein the nuclease cuts the genome at the cut site in the cell, and the donor nucleic acid guides the repair of the genome sequence by the modified sequence through the increased HDR rate.For example, the homology-directed repair (HDR) rate is increased by at least 1.5 times.In some cases, the homology-directed repair (HDR) rate is increased by at least 2 times.

[0023] In some aspects, the lower temperature is between 28°C and 32°C. Optionally, the lower temperature is between 30°C and 33°C. For example, the cells are grown at the lower temperature for at least 24 hours, or at least 48 hours, e.g., 1 to 5 days (1 day, 2 days, 3 days, 4 days, or 5 days). Optionally, the cells are grown at 37°C after the temperature shift.

[0024] In some aspects, the cell is a eukaryotic cell, such as a mammalian cell. In certain embodiments, the cell is a stem cell, such as an induced pluripotent stem cell (iPSC). In another specific embodiment, the cell is a primary cell. In another specific embodiment, the cell is a plant cell.

[0025] In some aspects, the nuclease used in the present invention includes all DNA sequence-specific endonucleases or RNA-guided DNA endonucleases. Optionally, the nuclease is a CRISPR nuclease selected from Cas nuclease or Cpf1 nuclease. For example, the nuclease is a Cas9 nuclease. For example, the CRISPR nuclease (e.g., Cas9) is introduced into cells in DNA format (e.g., DNA encoding Cas9 nuclease and sgRNA) or in RNA format (e.g., sgRNA / Cas9RNP or sgRNA / Cas9mRNA) together with sgRNA. Optionally, the sgRNA is synthetic and chemically modified. In some aspects, the donor nucleic acid contains symmetric homology arms. Optionally, the donor nucleic acid is complementary to the DNA strand in the genome that is cut by the nuclease.

[0026] In one aspect, the nuclease used in the present invention is a zinc finger nuclease (ZFN). In another aspect, the nuclease used in the present invention is a TALE nuclease (TALEN).

[0027] In certain embodiments, the present invention provides isolated cells produced by the above methods.

[0028] In certain embodiments, the present invention provides a pharmaceutical composition comprising the isolated cells produced by the above-described methods.

[0029] In one embodiment, the present invention provides a method for providing a protein of interest to a subject in need thereof, the method comprising: (a) introducing a donor nucleic acid encoding the protein of interest into a cell according to the methods described above; and (b) introducing the cell into the subject to express the protein of interest in the subject.

[0030] In one embodiment, the present invention provides a method for increasing the efficiency of homology-directed repair (HDR) in a cell's genome, comprising: introducing into the cell (i) a nuclease; and (ii) a donor nucleic acid comprising a symmetric homology arm, complementary to the DNA strand in the genome cleaved by the nuclease, and comprising a modified sequence to be inserted into the genome at a distance of more than 10 base pairs from the cleavage site; wherein the nuclease cleaves the genome at the cleavage site in the cell, and the donor nucleic acid guides the repair of the genome sequence with the modified sequence by an increased HDR rate. For example, the homology-directed repair (HDR) rate is increased by at least 1.5 times, or at least 2 times. Optionally, the method further comprises subjecting the cell to a temperature shift from 37°C to a lower temperature (for example, between 28°C and 35°C, or between 30°C and 33°C). For example, the cells are grown at the lower temperature for at least 24 hours, or at least 48 hours, eg, 1 to 5 days (1 day, 2 days, 3 days, 4 days, or 5 days).

[0031] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0032] "Nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer in either linear or circular, and in either single- or double-stranded form.

[0033] The terms "polynucleotide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0034] A "zinc finger DNA-binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0035] A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are responsible for binding of the TALE to its cognate DNA sequence. A single "repeat unit" (also called a "repeat") is typically 33-35 amino acids in length and shows at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins.

[0036] The term "CRISPR / Cas9 system" or "Cas9 system" refers to a system that can modify target nucleic acid through one of many DNA repair pathways. In some embodiments, the Cas9 system described herein promotes the repair of target nucleic acid through HDR pathway. In some embodiments, the Cas9 system comprises a gRNA molecule and a Cas9 molecule. In some embodiments, the Cas9 system further comprises a second gRNA molecule.

[0037] As used herein, "Cas9 molecule" or "Cas9 nuclease" refers to a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A "Cas9 polypeptide" is a polypeptide that can interact with a gRNA molecule and, in cooperation with the gRNA molecule, localize to a site containing a target domain, and in some embodiments, to a PAM sequence. Cas9 molecules include both native Cas9 molecules, engineered, altered, or modified Cas9 molecules, and Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference Cas9 sequence, e.g., a native Cas9 molecule. The term "altered, engineered, or modified" as used in this context simply refers to a difference from a reference or native Cas sequence and does not impose a restriction to a particular process or origin. Cas9 molecules can be nucleases (enzymes that cleave both strands of double-stranded nucleic acids) or nickases (enzymes that cleave one strand of double-stranded nucleic acids).

[0038] As used herein, "gRNA molecule" or "gRNA" refers to a guide RNA that can target a Cas9 molecule to a target nucleic acid. In one embodiment, the term "gRNA molecule" refers to a guide ribonucleic acid. In another embodiment, the term "gRNA molecule" refers to a nucleic acid that encodes a gRNA. In one embodiment, the gRNA molecule is non-naturally occurring. In one embodiment, the gRNA molecule is a synthetic gRNA molecule. In another embodiment, the gRNA molecule is chemically modified.

[0039] "Template nucleic acid," "donor nucleic acid," or "donor polynucleotide" refers to a nucleic acid sequence that can be used in combination with a nuclease (e.g., a Cas9 molecule) to alter the structure of a target location. In one embodiment, the template nucleic acid is modified, typically at or near the cleavage site, to have some or all of the sequence of the template nucleic acid. In one embodiment, the template nucleic acid is single-stranded. In an alternative embodiment, the template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, e.g., double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded DNA. In one embodiment, the template nucleic acid is RNA, e.g., double-stranded RNA or single-stranded RNA. In one embodiment, the template nucleic acid is an exogenous nucleic acid sequence. In another embodiment, the template nucleic acid sequence is an endogenous nucleic acid sequence, e.g., an endogenous homologous region. In one embodiment, the template nucleic acid is a single-stranded oligonucleotide corresponding to the plus strand of a nucleic acid sequence. In another embodiment, the template nucleic acid is a single-stranded oligonucleotide corresponding to the minus strand of a nucleic acid sequence.

[0040] " Homologous recombination repair " or " HDR " refers to the process of repairing DNA damage in cells using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid). Classical HDR typically operates when there is a critical excision at double-strand break, and forms at least one single-stranded portion of DNA. In normal cells, HDR typically involves a series of steps, such as break recognition, break stabilization, excision, single-strand DNA stabilization, DNA cross-over intermediate formation, cross-over intermediate disassembly, and ligation.

[0041] "Non-homologous end joining" or "NHEJ" refers to ligation-mediated repair and / or non-template-mediated repair, including classical NHEJ (cNHEJ), alternative NHEJ (altNHEJ), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0042] "Recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture by homologous recombination. "Homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells via the homology-directed repair mechanism. This process requires nucleotide sequence homology and uses a "donor" molecule to template repair of the "target" molecule, resulting in the transfer of genetic information from the donor to the target. This transfer may involve mismatch correction of heteroduplex DNA that forms between the disrupted target and the donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. This 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.

[0043] In the disclosed methods, the nucleases described herein create a double-stranded break in a target sequence (e.g., cellular chromatin) at a predetermined recognition site, and a "donor" polynucleotide having homology to the nucleotide sequence of the break region can be introduced into the cell. The presence of the double-stranded break has been shown to promote repair of the genomic sequence by the donor polynucleotide. The donor polynucleotide can be physically integrated, or alternatively, the donor polynucleotide can be used as a template for repair of the break via homologous recombination, where all or part of the nucleotide sequence, as in the donor, is introduced into the cellular chromatin. Thus, a first sequence in the cellular chromatin can be altered, and in certain embodiments, converted to a sequence present in the donor polynucleotide (referred to herein as a "modified sequence"). Thus, the use of the terms "replace" or "substitution" can be understood to refer to the replacement of one nucleotide sequence with another, and does not necessarily require the physical or chemical replacement of one polynucleotide with another nucleotide.

[0044] II. Nucleases The method of the present invention utilizes a nuclease to cut the genome of a cell, so that a template nucleic acid (transgene) guides the repair of the genome sequence in a targeted manner. In some embodiments, the nuclease is natural. In other embodiments, the nuclease is non-natural, for example, an engineered or modified version of a natural wild-type nuclease.

[0045] Nucleases include, but are not limited to, Cas proteins, DNA sequence-specific endonucleases, RNA-guided DNA endonucleases (e.g., Cpf1), restriction endonucleases, meganucleases, homing endonucleases, TAL effector nucleases, and zinc finger nucleases. Exemplary nucleases include, but are not limited to, type I, type II, type III, type IV, and type V endonucleases. For example, the nuclease is a CRISPR nuclease (e.g., Cas nuclease or Cpf1 nuclease). In certain embodiments, the nuclease is Cas9, e.g., Cas9 cloned from or derived from bacteria (e.g., S. pyogenes, S. pneumoniae, S. aureus, or S. thermophilus).

[0046] In some embodiments, the nuclease is a CRISPR / Cas nuclease system. The CRISPR (clustered regularly interspaced short palindromic repeats) locus encodes the RNA component of the system, and the cas (CRISPR-associated) locus encodes protein (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60) constitutes the gene sequence of the CRISPR / Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage.

[0047] Type II CRISPR, one of the best-characterized systems, executes double-strand breaks in target DNA in four sequential steps. First, two non-coding RNAs, the pre-crRNA and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNAs containing their respective spacers. Third, the mature crRNA:tracrRNA complex targets Cas9 to the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA adjacent to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Fourth, Cas9 mediates cleavage of the target DNA, creating a double-strand break within the protospacer.

[0048] In one embodiment, the Cas protein is a "functional derivative" of a native Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that shares qualitative biological characteristics with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence polypeptides and their fragments, provided that they share a biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or fragments thereof include, but are not limited to, mutants, fusions, and covalent modifications of a Cas protein or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, and derivatives of a Cas protein or fragments thereof can be obtained from cells, chemically synthesized, or obtained by a combination of these two procedures. The cells can be cells that naturally produce Cas proteins, or cells that naturally produce Cas proteins and have been genetically engineered to produce endogenous Cas proteins at higher expression levels, or to produce Cas proteins from exogenously introduced nucleic acids (which nucleic acids encode the same or different Cas proteins as the endogenous Cas). In some cases, the cells do not naturally produce Cas proteins but have been genetically engineered to produce Cas proteins.

[0049] In other embodiments, nuclease is zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN).ZFN and TALEN comprise heterologous DNA binding domain and cleavage domain.These molecules are well-known genome editing tools.See, for example, Gai, et al., Trends Biotechnol. 2013 July; 31(7): 397-405.

[0050] III.Host cells Any host cell for genome modification can be used in the present invention. The cell type can be a cell line or a natural (e.g., isolated) cell, such as a primary cell.

[0051] By way of example, suitable cells include eukaryotic (e.g., animal, plant, mammalian) cells and / or cell lines. Non-limiting examples of these cells or cell lines include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells. In certain embodiments, the cell line is a CHO, MDCK, or HEK293 cell line. Suitable cells also include stem cells, such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

[0052] IV. Delivery method Nucleases, nucleic acids encoding these nucleases, template nucleic acids, and compositions comprising the proteins and / or nucleic acids can be delivered by any suitable means to any cell type, in vivo or ex vivo.

[0053] Nuclease and / or donor construct described herein can also be delivered using a vector containing one or more sequences encoding ZFN, TALEN or CRIPSR / Cas system.Any vector system can be used, including but not limited to, plasmid vector, retrovirus vector, lentivirus vector, adenovirus vector, poxvirus vector, herpesvirus vector and adeno-associated virus vector.See also U.S. Patent No. 6,534,261;6,607,882;6,824,978;6,933,113;6,979,539;7,013,219;and 7,163,824 (all of which are incorporated herein by reference).In addition, it will be clear that any of these vectors can contain one or more sequences required for treatment. Thus, when more than one nuclease and donor construct are introduced into a cell, the nuclease and / or donor polynucleotide can be carried on the same or different vectors. When multiple vectors are used, each vector can contain sequences encoding one or more nucleases and / or donor constructs.

[0054] Conventional viral or non-viral gene transfer methods can be used to introduce the nucleic acid encoding the nuclease and donor construct into cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA or RNA plasmids, DNA MCs, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery into cells.

[0055] Non-viral delivery methods of nucleic acid include electroporation, lipofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA.Sonoporation, for example, using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acid.

[0056] Further exemplary nucleic acid delivery systems include systems provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam). (商標) and Lipofectin (商標) Cationic and neutral lipid polynucleotides suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424, WO 91 / 16024.

[0057] The preparation of lipid:nucleic acid complexes (including targeted liposomes, such as immunolipid complexes) is well known to those of skill in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); see U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0058] The use of RNA or DNA virus-based systems for delivery of nucleic acids encoding engineered ZFPs, TALEs, and / or CRISPR / Cas systems takes advantage of highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).

[0059] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing nucleases and / or donor constructs can also be administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA can be administered. Administration is by any of the routes typically used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering these nucleic acids are available and well known to those skilled in the art, and while more than one route can be used to administer a particular composition, certain routes can often provide a more immediate and effective response than another route.

[0060] It will be apparent that the nuclease-encoding sequence and the donor construct can be delivered using the same or different systems. For example, the nuclease and donor can be delivered by the same DNA MC. Alternatively, the donor polynucleotide can be delivered by the MC, while one or more nucleases can be delivered by a standard plasmid or AAC vector. Furthermore, different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration, and / or intramuscular injection). The vectors can be delivered simultaneously or in any sequential order.

[0061] The effect of genetic manipulation using the methods disclosed herein can be observed, for example, by Northern blotting of RNA (e.g., mRNA) isolated from the tissue of interest. Typically, if mRNA is present or the amount of mRNA is increased, it can be assumed that the corresponding transgene is expressed. Other methods for measuring the activity of the gene and / or the encoded polypeptide can be used. Different types of enzyme assays can be used, depending on the substrate used and the method for detecting the increase or decrease of reaction products or by-products. Furthermore, the level of the expressed polypeptide can be measured immunochemically, i.e., by ELISA, RIA, EIA, and other antibody-based assays well known to those skilled in the art, such as electrophoretic detection assays (either by staining or Western blotting).

[0062] V. Temperature shift The present invention encompasses subjecting host cells to a period of cold shock after introduction of the nuclease and / or donor nucleic acid. Cells can be shifted from 37°C to a lower temperature (cold shock) within minutes of transfection, or can be maintained at 37°C for a short period (e.g., 1 day) before being shifted to a lower temperature.

[0063] The duration for which the cells are cold shocked can range from a few hours to several days. In one embodiment, the cells are cold shocked for 1 to 4 days. It will be apparent that the duration of the cold shock will also vary depending on the cell type into which the nuclease is introduced.

[0064] Furthermore, the temperature to which cells are cold shocked can be any temperature that reduces cell division but at which nucleases are expressed and / or activated. Suitable temperatures will vary depending on the host cell type. For mammalian cells, cold shock temperatures include, but are not limited to, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, and even lower. Furthermore, the temperature can be varied during the cold shock period, provided that it remains low enough that cells are not dividing or are dividing at a reduced rate.

[0065] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. [Example]

[0066] Example 1 Cold shock increases the frequency of homologous recombination repair for gene editing in induced pluripotent stem cells Introduction One of the most promising applications of Clustered Regularly Spaced Palindromic Repeats (CRISPR) technology is its use in creating genetic models of human disease. CRISPR technology can be used on induced pluripotent stem cells (iPSCs) isolated from normal individuals to study disease phenotypes, or on iPSCs derived from diseased patients to revert putative disease-causing mutations to wild-type. The relative robustness of the CRISPR approach compared to zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has enabled empirical data generated by genome-wide association studies to be used to test protein-coding mutations as well as other non-coding mutations (3, 4). Despite many successes, gene editing in iPSCs has been hampered by the fact that homology-directed repair (HDR), the process by which exogenous donor DNA repairs CRISPR-induced double-strand breaks, is less efficient than in transformed cancer cell lines (5-8).

[0067] To overcome low HDR rates, researchers have employed several strategies, such as including antibiotic resistance genes on CRISPR plasmids and / or donor DNA (9). While effective, these strategies still leave undesired insertions of foreign DNA into the genome. Combining positive selectable markers with technologies that allow for excision of the selectable marker, such as the Cre / lox system or footprint-free PiggyBAC transposons, represents a significant improvement but extends the timeline, as clonal selection becomes a two-step process (2, 10). Methods using single-stranded oligonucleotide (ssODN) donor molecules avoid the problems of random integration and the presence of larger double-stranded DNA molecules in terms of unwanted "footprints," but are still susceptible to relatively low rates of successful repair and sequence conversion around the double-strand break site (7, 11). To overcome the difficulty of isolating rare clones, Miyaoka et al. devised a strategy to enrich for extremely rare clones using droplet digital PCR, clonal pooling, and sib selection (12). An additional strategy to increase HDR rates involves timing the delivery of the Cas9RNP complex to the nuclease by inducing cell cycle synchronization with a known chemical inhibitor of cell cycle progression (13). Here, a significant increase in HDR of up to 38% was achieved in synchronized HEK293 cells, whereas the effect of synchronization was minimal in primary human fibroblasts or H9 human embryonic stem cells. Specifications of ssODN structure and composition have also been shown to affect HDR rates. Lin et al. found that oligos with homology arms of at least 60 nucleotides were most effective, but strand complementarity was not a factor. A more detailed study by Richardson et al. of how donor oligo structure affects HDR in HEK293 cells used insights gained from in vitro binding of Cas9RNP-dsDNA complexes.Using a GFP reporter assay, they showed that asymmetric donor oligos, which are shorter with respect to the PAM site and complementary to the (+) strand (i.e., the non-target strand), are more effective at promoting HDR than symmetric donor oligos (14). Paquet et al.'s study optimizing HDR in iPSCs showed that efficient insertion of a desired mutation can be achieved with a (-) oligo complementary to the target strand, and that the frequency of integration of the desired mutation is distance-dependent from the CRISPR cleavage site. HDR fidelity could also be increased by introducing silent base changes into the oligo to disrupt the CRISPR recognition sequence (15).

[0068] We systematically evaluated gene editing steps in iPSCs to determine the best combination of delivery, CRISPR mode, and donor oligo design. We then tested the effect of a moderate "cold shock" on the cells' ability to perform HDR. Our optimized method uses a novel combination of Cas9-encoding mRNA, a symmetric donor oligo (-) complementary to the target strand, and lipids designed to deliver large RNA molecules combined with silent alterations to prevent re-editing, allowing for the desired genetic modifications to be introduced into the genome with 10-30% efficiency. Further exposure of cells to a short "cold shock" at 32°C can increase the amount of complete HDR by 2-10-fold, even when low efficiency repair is observed at 37°C.

[0069] Methods and Materials 1) Cell lines and cell culture Human mc-iPSCs were obtained from System Biosciences (SC301A-1) and maintained on Matrigel (BD Bioscience)-coated plates in mTeSR medium (Stem Cell Technologies) and 50 units / ml penicillin-streptomycin (Thermo Fisher Scientific) with daily medium changes (Ludwig, TE, et al. (2006). "Feeder-independent culture of human embryonic stem cells." Nat Methods 3(8): 637-646). For subculture, cells were washed with PBS and treated with Accutase (Thermo Fisher Scientific) for 5 minutes at 37°C. Cells were resuspended in mTeSR medium and centrifuged at 80g for 5 minutes. The cell pellet was replated in mTeSR medium supplemented with 10 μM ROCK inhibitor Y-27632 (Cayman Chemical).

[0070] 2) CRISPR and Cas9 Reagents CRISPR guide RNAs were designed using the Doench algorithm (http: / / portals.broadinstitute.org / gpp / public / ) and the Zhang Laboratory's CRISPR design tool (http: / / crispr.mit.edu). Guide sequences were subcloned into the plasmid pX458 (GenScript) or synthesized as IVTsgRNA (Thermo Fisher Scientific). GeneArt (登録商標) Platinum (登録商標)Cas9 nuclease was obtained from Thermo Fisher Scientific, and Cas9 mRNA (5meC, Ψ) was obtained from TriLink BioTechnologies. Repair templates (Ultramer, IDT) were designed as single-stranded oligonucleotides (ssODNs) with the target mutation in the center of the oligonucleotide and homologous genomic flanking sequences on either side of the mutation (Miyaoka, Chan et al. 2014, Richardson, Ray et al. 2016). Some ssODN designs also introduced silent mutations in the guide RNA binding sequence and PAM site. PCR primers were designed using Primer 3, and the primers were purchased from Sigma. See Table 1 for primer, probe, and oligonucleotide donor sequences. [Table 1-1] [Table 1-2]

[0071] 3) Transfection For lipid-based transfection of Cas9 mRNA and IVTgRNA in mc-iPSCs, the procedure was similar to that for IVTgRNA and Cas9 protein transfection with minor modifications (see Additional Methods). Specifically, 480 ng of IVTgRNA and 2 μg of Cas9 mRNA were first mixed in 50 μl of OptiMEM medium, followed by 2.5 μl of mRNA-InStem or Edit-Pro. (登録商標) (MTI-GlobalStem) was added. For homologous recombination repair experiments, various amounts of ssODN were added to the complex before adding lipid. To monitor transfection efficiency, 100ng of GFP mRNA was also added to each mixture. Plates were incubated in a 5% CO2 incubator at 37°C for 48 hours, and then cells were harvested for genomic DNA extraction.

[0072] 4) Genomic DNA extraction and PCR amplification of the edited region For genome extraction from transfected cells, the medium from each well was aspirated, and the cells were treated with 250 μl of Accutase (Thermo Fisher Scientific) for 10 minutes at 37°C. 750 μl of mTeSR medium was added to each well, and the cell suspension was transferred to a 1.5 ml Eppendorf tube and spun at 1000 g for 5 minutes. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN), and 100 ng of genomic DNA was used for PCR using Q5 polymerase (NEB) and target-specific primers (Table 1). Specifically, PCR amplification of the CAMK2D locus was performed using primers Camk2D-F and Camk2D-R. PCR amplification of the TGFBR1 locus was performed using primers TGFβR1-F and TGFβR1-R. The thermocycler conditions were 1 cycle at 98°C for 30 seconds, 31 cycles at 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute, and 1 cycle at 72°C for 1 minute. The PCR reaction was finally maintained at 4°C.

[0073] 5) Next-generation sequencing and analysis PCR amplicons were cleaned for library preparation by removing high-molecular-weight (HMW) genomic DNA and residual primers in a two-step cleanup. HMW DNA was removed by adding 0.6 v / v AmpureXP beads (Beckman Coulter), and the clear supernatant was transferred to a new plate. Primers were removed by adding 0.2 v / v AmpureXP beads to the transferred supernatant, which was then placed on the magnet again until clear, and then the supernatant was discarded. The beads were washed twice with 80% EtOH, air-dried, and resuspended in 20 μl of water to elute the DNA. Products were monitored for size using a Tapstation HSD5000 (Agilent Technologies) and quantified using Qubit HS DNA (Invitrogen). The cleaned PCR products were used in a NexteraXT kit (Illumina), modified to use half the manufacturer's standard reagent volumes. Samples were uniquely indexed using Illumina's standard indexing kit, with up to 384 unique i5 / i7 combinations. Amplification was performed with the heated lid closed: 72°C for 3 minutes, 98°C for 1 minute, followed by 12–14 cycles of 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes and cooling to 4°C. Libraries were size-selected using the same AmpureXP bead protocol as above and eluted in 15 μl of water. Products were run on a Tapestation HSD1000 (Agilent) and quantified by qPCR using the KAPA Library Quantification Kit for ABI (Kapa Biosystems). Libraries were normalized to 4 nM each in TE pH 8.0 and pooled in appropriate volumes according to the KAPA Library Quantification Data Analysis Template for Illumina (Kapa Biosystems). Following standard Illumina protocols, the library was denatured, diluted to 12 pM, and 1% v / v PhiX control was added.The run parameters were set to 150bp paired-end, 8bp dual index, using the MiSeq300v2 Reagent Kit (Illumina). Samples were demultiplexed using MiSeq Reporter v2.6 or bcl2fastq v2.17. Coverage at the guide sites after read deduplication was set to approximately 300x for clonal samples and at least 3000x for evaluation of diverse non-clonal populations.

[0074] NSD data analysis was performed using a proprietary pipeline. Briefly, quality filtering was performed on paired-end reads using PRINSEQ. The filtered reads were then aligned to the reference genome using BWA and then realigned using ABRA (assembly-based realigner) to improve indel detection. For quality assurance, the coverage depth of the amplicon was examined, and the entire amplicon region was examined for insertion and deletion frequencies. To calculate the indel frequency of CRISPR sites, the sgRNA sequence (18-20 bases) was used as the target window, and the number of wild-type and indel reads spanning this window was counted. Furthermore, indel reads were required to have at least one inserted or deleted base within this window, while wild-type reads had no indels within this window, ignoring point mutations. In addition to the overall percentage of indels, the percentage of in-frame indels was calculated to assess the disruptiveness of indels (Mose, LE, et al. (2014). "ABRA: improved coding indel detection via assembly-based realignment." Bioinformatics 30(19): 2813-2815). Indel length histograms and all other charts were plotted using R. The frequency of point mutations in the sgRNA guide region and its flanking regions was also examined. In the homology-directed repair (HDR) project, oligotypes were classified to assess HDR efficiency.

[0075] 6) ddPCR assay to detect CAMK2D wild-type and mutant sequences Follow the manufacturer's instructions and install the QX200 (登録商標) A Droplet digital PCR System (Bio-Rad Laboratories, CA) was used. ddPCR assays for detecting the CAMK2D wild-type and mutant sequences were designed using Primer Express and ordered from Life Technologies (Life Technologies, CA, USA). ddPCR reactions were assembled using standard protocols as follows: ddPCR Supermix for Probe (without dUTP) (Bio-Rad Laboratories, CA, USA) was prepared with 160 ng of sample genomic DNA, 1 μl of 20× FAM assay and 1 μl of 20× VIC assay (1× CAMK2D-ddPCR Primer F and CAMK2D-ddPCR Primer R, 900 nM each, 1× probe, 250 nM each), and 5 units of the restriction enzyme BamHI-HF. (登録商標) The supermix was combined with 1000kJ of 1000kcal of 1000kJ ...

[0076] 7) "Cold shock" experiments in transfected cells One day before transfection, mc-IPSCs were seeded into 24-well plates as described in the transfection section and divided into four groups (P1-P4). Groups P1-P3 were kept at 37°C, while P4 was incubated at 32°C for 24 hours. Cells were then transfected with IVT gRNA / Cas9 mRNA and ssODN using Edit-Pro as described. After transfection, group P1 was kept at 37°C until harvesting, while the remaining groups were transferred to 32°C until harvesting, except for group P3, which was returned to 37°C 24 hours after transfection. Cells were harvested 48 hours later for genomic DNA isolation, and indel formation and HDR were measured by either ddPCR or NGS, as described.

[0077] 8) Additional transfection methods For lipid-based transfection of DNA in mc-iPSCs, cells were plated at 1 x 10 per well one day before transfection. 5 On the day of transfection, 1 μg of pX458-CRISPR DNA was diluted in 50 μl of OptiMEM medium, followed by 2 μl of DNA-In (登録商標) Stem transfection reagent (MTI-GlobalStem) was added. For homologous recombination repair experiments, various amounts of ssODN were added to the mixture before adding lipids. The samples were gently mixed and incubated at room temperature for 15 minutes. The entire mixture was then added dropwise to the cells. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours, and the cells were harvested for genomic DNA extraction.

[0078] For lipid-based transfection of IVTgRNA and Cas9 nuclease in mc-iPSCs, the procedure was similar to that for DNA transfection with minor modifications (Liang, X., et al. (2015). "Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection." J Biotechnol 208: 44-53). Specifically, 480 ng of IVTgRNA and 2 μg of Cas9 nuclease were first mixed in 50 μl of OptiMEM medium and kept at room temperature for 10 minutes to form a stable RNP complex. After that, 2.5 μl of mRNA-InStem or Edit-Pro (MTI-GlobalStem) was added. For homologous recombination repair experiments, various amounts of ssODN were added to the mixture before lipid addition. 100 ng of GFP mRNA was also added to each mixture to monitor transfection efficiency. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours, and the cells were harvested for genomic DNA extraction.

[0079] For nucleofection of pX458 CRISPR plasmid with or without ssODN, mc-iPSCs were first cultured in Matrigel-coated 10 mm dishes until they reached 60–70% confluence. Cells were washed with PBS and treated with 3 ml of Accutase (Thermo Fisher Scientific) at 37°C for 5–8 minutes until all cells were dissociated. Cells were resuspended in TeSR medium and counted. Cells were then transferred to a 15 ml tube and centrifuged at 80 g for 5 minutes. After removing the supernatant, cells were collected at a concentration of 1 × 10 7The cells were resuspended in P3 or P4 nucleofection solution (Lonza, Basel, Switzerland) at 1 / ml. 20 μl of the cell suspension was transferred to tubes, and 1 μg of pX458-CRISPR was added to each tube. For homologous recombination repair experiments, various amounts of ssODN were also added to the mixture. The suspension was then transferred to each well of an 8-well strip (Lonza, Basel, Switzerland), taking care to avoid creating bubbles, and then transferred to an Amaxa (登録商標) 4D-Nucleofector (登録商標) Electroporation was performed using a 24-well Matrigel-coated plate (Lonza, Basel, Switzerland) under program CM-113 or CE-118. Nucleofected cells were directly plated into individual wells of a Matrigel-coated plate containing 500 μl of prewarmed mTESR medium with 10 μM of ROCK inhibitor Y-27632 in each well. The plate was incubated at 37°C in a 5% CO incubator for 48 hours, after which the cells were harvested for genomic DNA extraction.

[0080] For nucleofection of IVTgRNA and Cas9 protein in mc-iPSCs, the procedure was similar to that for DNA nucleofection with minor modifications. Specifically, 480 ng of IVTgRNA and 2 μg of Cas9 protein were first mixed in OptiMEM medium to a final volume of 5 μl and kept at room temperature for 10 minutes to form a stable RNP complex. For homologous recombination repair experiments, various amounts of ssODN were also added to the mixture. The complex was then transferred to 20 μl of cell suspension in P3 or P4 nucleofection solution and transfected with Amaxa nucleofection solution as described above. (登録商標) 4D-Nucleofector (登録商標) (Lonza, Basel Switzerland) using the program CM-113 or CE-118.

[0081] result I. Efficient HDR in iPSCs using CRISPR / Cas9 RNA format and lipid delivery. To find the best conditions for generating HDR within the CAMK2D gene, we evaluated several aspects of gene editing protocols. First, we determined which CRISPR modality (e.g., all-in-one plasmid DNA, sgRNA and Cas9 mRNA, or sgRNA in vitro transcription (IVT) / Cas9 ribonucleoprotein) and delivery method (e.g., nucleofection or lipids formulated for enhanced delivery of large DNA and RNA molecules) produced the greatest number of double-strand breaks at two specific locations within the CAMK2D gene (Figure 1a), as detected by PCR amplicon next-generation sequencing (NGS). The best NHEJ-induced indel rates for each modality and delivery method are presented in Table 2. The complete matrix of conditions used to determine optimal indel formation was retested to determine the best combination of modality and delivery to promote HDR, and these are also presented in Table 2. A multiplex ddPCR assay was used to measure the incorporation of four base changes designed to disrupt the CRISPR recognition sequence and specific mutations designed to create a kinase-knockout version of CAMK2D on the same oligo (Figure 1b). The amount of wild-type, unedited sequence was determined using a different probe that specifically detects the non-HDR wild-type allele (Figure 1b). The donor oligo design was symmetric with respect to the length of the homology arms, and the CRISPR cut site was positioned as close as possible to the intended kinase knockout mutation. The donor sequence was also homologous to the non-target CRISPR cut strand (+). The four silent mutations introduced by the oligos altered the CRISPR recognition sequence of guide CAMK-CR2 at four positions and mutated the PAM sequence and introduced three sequence changes in guide CAMK-CR1. The assay was validated using both synthetic fragments of different DNA sequences in clones previously generated in HEK293 cells known to be heterozygous and homozygous for the HDR donor oligo sequences (data not shown). The best HDR rates for all conditions compared are presented in Table 2, and data for the best combinations are presented in Figure 2a.IVT sgRNA / Cas9mRNA and EditPro. (登録商標) For lipids, we confirmed that 9% of all alleles for CAMK-CR1 and 19% of alleles for CAMK-CR2 had incorporated the donor oligo sequence (Figure 2b). To confirm the ddPCR results, we performed next-generation sequencing (NGS) on PCR amplicons derived from transfected populations of iPSCs (Figure 2c). For IVT sgRNA / Cas9 mRNAs involving both CAMK-CR1 and CAMK-CR2, the desired base changes, which would indicate successful HDR into the locus, were observed at the precise genomic coordinates and at frequencies closely consistent with those determined by ddPCR, including two guanine substitutions that were not directly measured by the ddPCR assay. These two substitutions were more distal to the CRISPR cut site and observed at a lower frequency than silent mutations designed to disrupt sgRNA annealing. [Table 2]

[0082] II. "Cold shock" increases HDR rate. Based on our previous observation that T-antigen temperature-sensitive immortalized cell lines grown and maintained at 32°C underwent HDR more efficiently than similar cell lines grown at 37°C (data not shown), we tested whether exposing mc-iPSC cell lines to 32°C for various intervals affected HDR efficiency. The experimental design and the resulting percentage of alleles undergoing HDR, as measured by ddPCR at each temperature, are presented in Table 3. Using normal culture conditions at 37°C as the HDR baseline (group PL1), we observed HDR frequencies of 7.50% and 5.0% for guide CAMK-CR1 and 16.16% and 8.86% for guide CAMK-CR2 at concentrations of 10 pmol and 30 pmol, respectively. When cells were transferred to 32°C immediately after transfection, maintained there for 24 hours, and then transferred to 37°C for another 24 hours (group PL2), we observed a statistically significant increase in HDR of 1.8- to 2.3-fold. This effect was more pronounced at the 30 pmol concentration, where lower HDR efficiency was observed at baseline. Exposing cells to 32°C for 48 hours after transfection (group PL3) also had a statistically significant effect on HDR, increasing HDR by 2.0-3.6-fold, and this effect was even more pronounced under conditions where HDR was lower at baseline. [Table 3]

[0083] III. "Cold shock" and alternative single-stranded oligonucleotide donor designs affect the efficiency of HDR. Recent data suggest that precise donor oligo design can dramatically affect the efficiency of donor oligo HDR. More specifically, oligos with asymmetric lengths (shorter near the CRISPR cut site) and complementary sequences to the non-target strand (the strand not initially cleaved by Cas9) promote more efficient HDR than oligos symmetric around the CRISPR cut site and complementary to the target strand, as employed to edit the CAMK2D locus (14). To directly compare the two designs and further test the effect of cold shock on HDR, we designed a gene editing experiment to compare the amount of HDR observed with symmetric target strand oligo donors to the amount of HDR observed with asymmetric non-target strand oligo donors designed to introduce the same sequence changes (Figure 1b). HDR amounts were determined by amplicon-based NGS, and the resulting sequence data were analyzed in several ways: 1) the overall amount of HDR at the locus, i.e., the amount of oligo directed repair, regardless of whether all or part of the intended change is present; 2) the percentage of HDRs that represented “full HDR,” which is oligo recombination repair in which all six desired base changes are complete (% full oligos); 3) the percentage of HDRs that were once repaired but estimated to be re-edited due to the reintroduction of indels into the converted sequence (% edited oligos); and 4) HDR percentage (partial oligo%), where partial oligo recombinational repair occurred such that the sequence did not exhibit two more distal sequence changes (the CAMK2 kinase knockout mutations of interest) (Figure 3a and Table 4).

[0084] As initially observed by ddPCR, guide CAMK-CR2 was more efficient at promoting overall HDR than guide CAMK-CR1 under baseline conditions, i.e., when cells were transfected and maintained at 37°C, although the overall HDR was lower (Figures 2b and 3a). The amount of overall HDR across all temperature conditions and oligo concentrations was comparable to that of guide CAMK-CR1. Overall, statistically significant increases in overall HDR were observed across all comparisons of temperature, guide, and oligo design (Figure 3a and Table 4). For guide CAMK-CR1, the amount of overall HDR for both oligos was essentially the same across the three temperature conditions, and an approximately 2.9-fold increase in overall HDR was observed under the PL3 temperature condition for both oligo designs (Figure 3a). For CAMK-CR2, the overall fold increase in HDR relative to the symmetric oligo C-CR2 was approximately 2.4-fold, but the magnitude of the response was 40% for alleles that experienced some HDR (Figure 3a). For the asymmetric design C-CR2, a 3.5-fold increase in HDR was observed between PL1 and PL3, although the magnitude of the response was approximately half that observed with the symmetric guide (Figure 3a). However, when considering only the amount of "full" HDR observed as a result of "cold shock," the type of oligo used had a dramatic effect, particularly for guide CAMK-CR2, where the difference in overall HDR between the first two designs was greater, with the symmetric oligo design superior in directing the conversion of all six nucleotide changes (Figure 3b and Table 4). For guide CAMK-CR1, the amount of overall HDR was similar for both oligo types across all temperature conditions, and the amount of "full" HDR was also greater for the symmetric oligo, although the difference was statistically significant only under PL3 conditions (Figure 3b and Table 4). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0085] To extend these findings to other loci and further test the effects of "cold shock" and oligo design on HDR, we designed gene editing experiments to insert silent changes and SNPs into the TGFRB1 locus. The locations of the two guides tested are shown in Figure 4a, and the sequences of the four donor oligos, the locations of the desired sequence changes, and their relationship to the guide locations are shown in Figures 4b and 4c. Guide TR-CR2 was designed to induce cleavage approximately 31 bp 3' of the desired A-to-C sequence change (Figure 4b). Both the symmetric and asymmetric donor oligos also contained three additional sequence changes designed to prevent guide recognition and re-editing at the locus. The lengths of the homology arms are also listed in Figure 4b. Guide TR-CR3 was designed to induce cleavage approximately 30 bp 3' of the desired C-to-T sequence change (Figure 4c). Similar to TR-CR2 and its ssODN, three additional silent sequence changes were also included to prevent re-editing of the converted locus. The length of the homology arms was designed to be as close as possible to the ssODN used for guide TR-CR2 (Fig. 4c).

[0086] Using the IVTsgRNA / Cas9RNA lipid format, both CRISPRs were efficient at generating indels in mc-iPSC lines; in the absence of repair oligos, the percentage of alleles with indels, as determined by NGS, was 92% for TR-CR2 and 64% for TR-CR3. In the presence of both repair oligos, guide TR-CR2 led to highly efficient HDR overall rates of 60% for the symmetric guide TR-CR2 and 42% for the asymmetric design in condition PL1 at 37 °C (Figure 5A and Table 5). For guide TR-CR3, HDR overall percentages of 41% and 34% were observed at 37 °C for the symmetric and asymmetric designs, respectively (Figure 5A and Table 5).

[0087] As observed with CAMK2D, culturing cells at 32°C for either 24 hours (group PL2) or 48 hours (group PL3) resulted in increased HDR; however, the effect was generally minimal given the relatively high HDR rates initiated at 37°C and, in most cases, did not reach statistical significance (Figure 5a and Table 5). However, the "cold shock" effect was more pronounced with asymmetric oligos, in which the amount of "full HDR" at 37°C was lower than with symmetric oligos. Here, for guide TR-CR2 and asymmetric donor T-CR2, statistical significance was achieved when comparing the amount of total HDR observed at 37°C (PL1) with the amount observed when cells were cultured at 32°C for 48 hours (PL3). For guide TR-CR3 and asymmetric guide T-CR3, statistical significance was achieved in both PL2 and PL3 conditions (Figure 5a and Table 5). However, when considering only the amount of "complete" HDR observed as a result of "cold shock" and baseline conditions, the type of oligo used had a dramatic effect (Fig. 5b and Table 5). In all comparisons except one (TR-CR2 / PL3), symmetric donor oligos were statistically significantly better than their asymmetric counterparts at directing "complete" HDR repair (Fig. 5b and Table 5). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0088] IV. The lower the base HDR rate, the more effective the "cold shock" is. To investigate whether "cold shock" is effective in cell types other than the specific mc-iPSC line under study, we repeated the same CAMK2D gene editing experiments used to derive the data in Table 3 in HEK293 cells, determining HDR levels using ddPCR (Table 6) and specific HDR categories by NGS (Figures 6 and 7). Overall baseline HDR levels for both CAMK2D sgRNAs and the two donor oligo concentrations were approximately 1% at 37°C (Tables 6, 7, and Figure 6). These results were obtained using both a plasmid-based all-in-one CRISPR format (data not shown) and an IVT sgRNA / Cas9 RNA format used in iPSCs in an independent experiment. This contrasts with what was observed in the mc-iPSC line, where overall HDR levels exceeded 10% and 20% for CAMK-CR1 and CR2 at 10 pmol concentrations, respectively, despite the HEK293 cell line being transfected with the same relative efficiency (Figure 3a and data not shown). The extent to which locus and cell type can determine the level of gene editing has been described by others (8). However, despite the low HDR rates observed at 37°C, "cold shock" under both 24-hour (PL2) and 48-hour (PL3) conditions resulted in a statistically significant 6.9-fold increase in overall HDR for the best sgRNA, CAMK-CR2, as determined by ddPCR in both the 24-hour and 48-hour conditions (Table 6). Both guides and all conditions resulted in a statistically significant increase in overall HDR in response to "cold shock" (Figure 6 and Table 7).

[0089] Amplicon-based NGS and analysis confirmed that "cold shock" resulted in a statistically significant increase in overall HDR for both guides, except for the comparison of PL1 vs. PL3 for guide CAMK-CR2. Overall, the increase in "total" HDR exceeded 5-20 fold for both guides and all conditions (Figure 6 and Table 7). [Table 6] [Table 7-1] [Table 7-2] [Table 7-3]

[0090] V. "Cold shock" does not affect the expression of pluripotency markers To test whether exposing mc-IPSCs to a period of low temperature could affect their differentiation potential into various cell lineages, we performed the same "cold shock" protocol used to determine whether this process affected HDR rates and stained the cells with antibodies that recognize protein antigens whose expression is indicative of pluripotency. The results of these studies demonstrate that the expression of the markers SSEA3, Nanog, and OCT4 is unchanged as a result of exposing cells to 32°C for either 24 or 48 hours (Figure 7a-c).

[0091] VI. "Cold shock" is effective in increasing HDR rates under various temperature conditions. To test the effect of exposing cells to temperatures lower than 32°C, we repeated the cell culture protocol described above and exposed mc-IPSCs to either 30°C, 28°C, or 32°C for both 24 and 48 hours.

[0092] The resulting PCR amplicons were analyzed by both ddPCR and NGS (Table 8, Table 9, and Figure 8). In general, the increase in overall HDR (Table 9 and Figure 8) and the increase in "full" HDR (Figure 8) were comparable across the three temperatures tested, and these data indicate that the increase in HDR is independent of the temperature conditions at 32°C. [Table 8] [Table 9-1] [Table 9-2] [Table 9-3]

[0093] Consideration The rapid development of CRISPR-based genome engineering methodologies necessitates an agnostic and systematic evaluation process to maximize the benefits from this technology. Here, we report an optimized CRISPR modality / delivery combination that is highly effective in promoting HDR in mc-iPS cells. We then used this method to evaluate whether exposure to lower temperatures could enhance HDR efficiency and found that exposure to 32°C for 24 or 48 hours, or "cold shock," could increase HDR rates by more than twofold. Given that considerable efforts have been made to find ways to increase HDR rates, including chemical inhibition of DNA repair enzymes to "drive" the repair process from non-homologous end joining to HDR (16) and blocking and synchronizing cells at the G2 / M boundary with other inhibitors (13), our method offers a more "physiological" approach that may have broader applications, especially when gene editing is applied in therapeutic settings.

[0094] Interestingly, the "cold shock" effect is more dramatic when lower HDR rates (1–20% of alleles) are observed and decreases as the base HDR rate increases beyond 30%. This suggests a theoretical limit, at least for the number of alleles that can be altered by this approach. The exact mechanism by which "cold shock" increases HDR is currently under investigation. A mechanism similar to that used by zinc finger nucleases to increase indel formation may be promising (17). The effect of "cold shock" on indel formation using highly efficient CRISPRs was observed to be minimal, as observed with the CAMK2D-guided CAMK-CR1 and TGFBR1-guided TR-CR2 loci. Conversely, the increase in cleavage efficiency and indel formation is more pronounced when cleavage efficiency and indel formation are lower, as observed at the CAMK2D locus in HEK293 cells. While increased indel formation may clearly contribute to higher HDR rates, it does not explain all of the increases observed with "cold shock" or why "full HDR" is favored under low-temperature conditions. One possible mechanism that could contribute to the increased HDR rate is that growing cells at 32°C affects the cell cycle, causing more cells to accumulate in G2 / M; however, our initial findings so far have not shown any cell cycle effects to support this hypothesis (data not shown). A third, and more likely contributing, factor is that low temperature has a thermodynamic effect that acts to stabilize recombination intermediates. Studies are currently underway to understand the mechanism in detail. One potential concern is that "cold shock" may have a negative effect on pluripotency. Preliminary analyses looking at three standard markers of pluripotency, Oct4, SSEA3, and Nanog (18, 19), suggest this is not an issue, although some loss of Nanog expression may occur with prolonged exposure to low temperatures (Figure 7). Clearly, more research is needed to ensure that "cold shock" is effective and generalizable across cell lines and applications.

[0095] We also show that the structure of the donor oligo used to promote HDR can significantly affect both the overall frequency and type of HDR that occurs. At the two loci tested, both symmetric target-strand and asymmetric non-target-strand oligo designs were able to induce high levels of overall HDR, but symmetric target-strand oligos induced "full HDR" more efficiently than asymmetric non-target-strand oligos, especially under "cold shock" conditions. While these data are inconsistent with those of Richardson et al. (14), our data are consistent with those of Paquet et al. (15), in which donor oligos were designed for the same strand.

[0096] In summary, we have developed a protocol for gene editing in iPSCs that does not require the use of nucleofection or selection to obtain a cell population in which directed genome sequence changes have been efficiently generated by the HDR process. We also show that HDR can be effectively increased by simply introducing brief, physiological exposure to lower temperatures, which will be broadly useful across many genome engineering applications.

[0097] References TIFF2026012785000021.tif228150

Claims

1. 1. A method for increasing the efficiency of homology directed repair (HDR) in the genome of a cell, comprising: (a) introducing into a cell: (i) a nuclease; and (ii) a donor nucleic acid comprising a modified sequence to be inserted into the genome; and (b) subjecting the cells to a temperature shift from 37°C to a lower temperature; Including, A method in which a nuclease cleaves the genome at the cleavage site in a cell, and a donor nucleic acid directs repair of the genomic sequence with a modified sequence by an increased HDR rate.

2. 2. The method of claim 1, wherein the lower temperature is between 28°C and 35°C.

3. 2. The method of claim 1, wherein the lower temperature is between 30°C and 33°C.

4. 10. The method of claim 1, wherein the cells are grown at said lower temperature for at least 24 hours.

5. The method of claim 1 , wherein the cell is a mammalian cell.

6. 6. The method of claim 5, wherein the cells are selected from stem cells, induced pluripotent stem cells (iPSCs), or primary cells.

7. 2. The method of claim 1, wherein the nuclease is a CRISPR nuclease selected from a Cas nuclease or a Cpf1 nuclease.

8. 8. The method of claim 7, wherein the nuclease is a Cas9 nuclease.

9. 8. The method of claim 7, wherein the CRISPR nuclease is introduced into the cell together with the sgRNA in either DNA or RNA format.

10. 10. The method of claim 9, wherein the sgRNA is synthetic and chemically modified.

11. 10. The method of claim 9, wherein DNA encoding the Cas9 nuclease and the sgRNA is introduced into the cell.

12. 10. The method of claim 9, wherein the sgRNA / Cas9RNP is introduced into the cell.

13. The method of claim 9, wherein sgRNA / Cas9 mRNA is introduced into the cell.

14. 2. The method of claim 1, wherein the donor nucleic acid contains symmetric homology arms and is complementary to the DNA strand in the genome that is cleaved by the nuclease.

15. 2. The method of claim 1, wherein the rate of homology directed repair (HDR) is increased by at least 1.5 fold.

16. A cell produced by the method of claim 1.

17. A pharmaceutical composition comprising the cells of claim 16.

18. 1. A method for providing a protein of interest to a subject in need thereof, comprising: (a) introducing into a cell a donor nucleic acid encoding a protein of interest according to the method of claim 1; and (b) introducing the cells into a subject to express the protein of interest in the subject; A method comprising:

19. 1. A method for increasing the efficiency of homology-directed repair (HDR) in the genome of a cell, comprising: introducing into the cell (i) a nuclease and (ii) a donor nucleic acid comprising symmetric homology arms, complementary to a DNA strand in the genome that is cleaved by the nuclease, and comprising a modified sequence to be inserted into the genome more than 10 base pairs away from the cleavage site; wherein the nuclease cleaves the genome at the cleavage site in the cell; and the donor nucleic acid directs repair of the genomic sequence with the modified sequence by an increased HDR rate.

20. 20. The method of claim 19, further comprising subjecting the cells to a temperature shift from 37°C to a lower temperature.

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