Method for producing genome-edited cells and kit therefor

Abstract

A method for producing a genome-edited cell is provided, the method comprising synchronizing the cell cycle of a cell, treating the cell with an agent that alters a DNA repair pathway, introducing donor DNA into the cell, and inducing a sequence-specific double-strand break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-strand break is repaired according to all or part of the donor DNA sequence by a homology-directed repair pathway or an equivalent pathway, resulting in a genome-edited cell.

Description

[Technical field]

[0001] The present invention relates to a method for producing genome-edited cells and a kit therefor. More specifically, the present invention relates to a method for producing genome-edited cells, a method for editing the genome of a cell, a method for detecting gene editing on one or both alleles, and a kit for genome editing. The present invention claims priority to U.S. Provisional Patent Application No. 62 / 978,312, filed February 19, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Human induced pluripotent stem (iPS) cells have been widely employed to study human diseases, including genetic disorders, due to their ability to maintain a normal diploid karyotype through serial passages and differentiate into multiple derived cell types. In general, gene modeling consists of modifying a target site by either deleting, inserting, or replacing a specific DNA sequence. More specifically, high-precision gene editing aims to make modifications in the genome to correct or re-create a pathogenic variant at a single base position. The CRISPR-Cas9 system is the most used tool to create targeted DNA double strand breaks (DSBs) in the genome that are subsequently repaired by endogenous cellular DNA DSB repair pathways. Primarily, nonhomologous end joining (NHEJ) results in insertion and deletion (indel) mutations, while microhomology-mediated end joining (MMEJ) causes predictable deletions. NHEJ and MMEJ are collectively referred to as mutagenic end joining (MutEJ) because both repair outcomes can result in the loss or gain of DNA sequences.To generate single base changes and other designed modifications, the homology directed repair (HDR) pathway is utilized in combination with customized single- or double-stranded DNA repair templates, such as PCR fragments, donor plasmids, or single stranded donor oligonucleotides (ssODNs). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Howden SE, et al., A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells, Stem Cell Reports, 7(3), 508-517, 2016. [Non-Patent Document 2] Alewo Idoko-Akoh, et al., High fidelity CRISPR / Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells, Sci Rep, 8(1):15126, 2018. Summary of the Invention [Problem to be solved by the invention]

[0004] However, recent approaches have been limited in efficiency and applicability, primarily due to the lack of direct selection and the dominance of MutEJ outcomes over precise repair (see, for example, non-patent literature 1 and 2).

[0005] The present invention aims to provide a technology for efficiently producing genome-edited cells in which targeted DNA double-strand breaks (DSBs) in the genome are repaired by the homology-directed repair (HDR) pathway or an equivalent pathway (alternative pathway). [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A method for producing a genome-edited cell, comprising: synchronizing a cell cycle of the cell; treating the cell with an agent that alters a DNA repair pathway; introducing donor DNA into the cell; and inducing a sequence-specific double-stranded break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, thereby obtaining the cell whose genome has been edited. [2] The method of [1], wherein synchronizing the cell cycle of the cells comprises incubating the cells at 25°C to 35°C for 24 to 72 hours. [3] The method of producing according to [1] or [2], wherein synchronizing the cell cycle of the cell comprises treating the cell with a Cell Division Cycle 7 (CDC7) inhibitor. [4] The method for producing according to any one of [1] to [3], wherein the agent that alters the DNA repair pathway comprises a DNA-PK inhibitor or a DNA ligase IV inhibitor. [5] The method for production described in any of [1] to [4], wherein the agent that alters the DNA repair pathway comprises a combination of a DNA-PK inhibitor and a DNA ligase IV inhibitor. [6] The method according to any one of [1] to [5], wherein the sequence-specific double-stranded cleavage is induced by a ribonucleoprotein (RNP) complex comprising Cas9 and gRNA. [7] The method according to any one of [1] to [6], wherein the donor DNA is a single-stranded donor oligonucleotide. [8] The manufacturing method described in [7], wherein the donor DNA has a nucleotide sequence that modifies a protospacer sequence or a protospacer adjacent motif (PAM) sequence. [9] The method according to any one of [1] to [8], wherein the donor DNA has a mutation, and a cell is obtained in which the mutation has been introduced into one or both alleles.

[10] The method of any one of [1] to [9], wherein the donor DNA comprises a combination of multiple types of donor DNA that correspond to the same region on the genome.

[11] The manufacturing method described in

[10] , wherein the donor DNA comprises a combination of a first donor DNA and a second donor DNA, the first donor DNA having a mutation of interest and the second donor DNA having a mutation that modifies a protospacer sequence or a PAM sequence, and the mutation of interest is introduced into only one allele, resulting in a cell in which the protospacer sequence or PAM sequence of the other allele is modified.

[12] The method according to any one of [1] to

[11] , wherein the cells are induced pluripotent stem (iPS) cells.

[13] A method for editing the genome of a cell, comprising: synchronizing a cell cycle of the cell; treating the cell with an agent that alters a DNA repair pathway; introducing donor DNA into the cell; and inducing a sequence-specific double-stranded break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by homologous recombination or an equivalent pathway based on all or a portion of the base sequence of the donor DNA, thereby obtaining the cell having an edited genome.

[14] A method for detecting genome editing on one or both alleles, comprising: introducing donor DNA into a cell, the cell having a gene encoding a fluorescent protein that emits a first fluorescence in both alleles, the donor DNA having a mutation that changes the first fluorescence of the fluorescent protein to a second fluorescence; inducing a sequence-specific double-stranded break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA to obtain a genome-edited cell; and analyzing the fluorescence of the genome-edited cell, whereby, if (i) a first fluorescence is detected and a second fluorescence is not detected, (i-1) both alleles of the fluorescent protein locus have not changed, or (i-2) one allele of the fluorescent protein locus has not changed and an insertion mutation or deletion mutation has occurred in the other allele. (ii) if the first fluorescence is not detected and the second fluorescence is detected, (ii-1) it is indicated that both alleles of the fluorescent protein locus have been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, or (ii-2) it is indicated that one allele of the fluorescent protein locus has been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, and an insertion mutation or deletion mutation has occurred in the other allele; (iii) if neither the first fluorescence nor the second fluorescence is detected, it is indicated that an insertion mutation or deletion mutation has occurred in both alleles of the fluorescent protein locus; and (iv) if both the first fluorescence and the second fluorescence are detected, it is indicated that one allele of the fluorescent protein locus has been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, and the other allele has not changed.

[15] The method according to

[14] , wherein the fluorescent protein that emits a first fluorescence is a green fluorescent protein (GFP), the fluorescent protein that emits a second fluorescence is a blue fluorescent protein (BFP), and the donor DNA has a mutation that replaces the 66th tyrosine residue of GFP with a histidine residue to modify it into BFP.

[16] A genome editing kit containing a CDC7 inhibitor, a DNA-PK inhibitor, and a DNA ligase IV inhibitor.

[17] The genome editing kit described in

[16] , further comprising a sequence-specific DNA cleaving enzyme and donor DNA.

[18] The genome editing kit described in

[17] , wherein the donor DNA has a mutation that replaces the 66th tyrosine residue of GFP with a histidine residue to modify it into BFP. Effect of the Invention

[0007] According to the present invention, a technique can be provided for efficiently producing genome-edited cells in which targeted DNA double-strand breaks (DSBs) in the genome are repaired by the homology-directed repair (HDR) pathway or an equivalent pathway. [Brief description of the drawings]

[0008] [Figure 1A] 1A-1E show visualization and quantification of DNA repair outcomes of a fluorescent DNA repair assay in human iPS cells. FIG. 1A shows the sequences of the GFP reporter target site (SEQ ID NO:60 and 61) and the designed modifications to the BFP. The sequence of gRNA GFPx199 (SEQ ID NO:62), GFP fluorescent residue p.Y66 (SEQ ID NO:60), and BFP fluorescent residue p.H66 (SEQ ID NO:64) are shown. Nucleotide changes are introduced into the ssODN sequence (SEQ ID NO:63) and the resulting BFP sequence product (SEQ ID NO:64).

[0009] [Figure 1B] FIG. 13 is a schematic diagram of the expected outcome of GFP editing in heterozygous AAVS1-CAG::EGFP (GFP) iPS cell lines and its distribution in FACS.

[0010] [Figure 1C] Confocal microscopy images of a non-edited heterozygous GFP iPS cell line (control) and the fluorescent conversion outcome after co-introduction of Cas9 RNP and Y66H ssODN repair template (RNP+ssODN). Scale bar is 100 μm. Representative images from three independent experiments with similar results are shown.

[0011] [Figure 1D] Representative FACS plots of unedited GFP iPS cells (top) and cells treated with Cas9 RNP and Y66H ssODN (bottom).

[0012] [Figure 1E] Figure 1 shows quantification of HDR, MutEJ or unmodified DNA repair outcomes by FACS analysis. Data are presented as mean ± SD of three independent technical replicates of electroporation for each condition.

[0013] [Figure 2A] 2A-2F show that cold shock and cell cycle synchronization using XL413 improve HDR efficiency in iPS cells. Figure 2A shows the experimental timeline (top) for no cold shock [A] and cold shock 24 h after electroporation (EP) on day 0 [B] or cold shock 48 h after electroporation (EP) [C]. EdU / PI staining was performed after 48 h of culture and FACS analysis was performed on day 8. The obtained effect on DNA repair outcome frequency is shown (bottom).

[0014] [Figure 2B]Figure 2 shows the ratio of HDR / MutEJ repair outcomes measured in Figure 2A. Statistical significance was calculated by unpaired two-tailed t-test, and P values ​​are indicated as **p<0.01, ***p<0.001. Cold shock significantly improved the HDR / MutEJ ratio.

[0015] [Figure 2C] Representative FACS plots of EdU / PI staining (top) and mean EdU intensity of S-phase cells (middle right) for cell cycle quantification 48 h after EP (middle left) without cold shock [A] or with 48 h cold shock after EP [C]. Schematic of the cell cycle showing cold shock arrest at G2 / M phase (bottom).

[0016] [Figure 2D] Experimental timeline of XL-413-induced cell cycle arrest 24 h after EP without [A] or with [C] cold shock (top). EdU / PI staining was performed after 24 h of culture and FACS analysis was performed on day 8. Repair outcomes quantified in the absence or presence of XL413 (bottom).

[0017] [Figure 2E] Figure 2D shows the ratio of HDR / MutEJ repair outcomes measured in Figure 2D. Statistical significance was calculated by unpaired two-tailed t-test, and P values ​​are indicated as ***p<0.001. Cold shock and XL413 treatment significantly improved the HDR / MutEJ ratio.

[0018] [Figure 2F] Representative FACS plots of EdU / PI staining after 24 hours of EP for untreated and XL413-treated cells under standard culture conditions [A] (top), as well as quantification of cell cycle (middle left) and mean EdU intensity of S-phase cells (middle right). Schematic diagram showing XL413-induced cell cycle arrest at early G1 / S (bottom). All data are presented as mean ± SD of three technical replicates for each treatment.

[0019] [Figure 3A] Figures 3A-J show that combining DNA repair regulation and cell cycle synchronization synergistically promotes HDR in iPS cells. Figure 3A shows a list of test compounds with their functions against molecular targets (left) and an experimental timeline of compounds before (4 hours) and after (48 hours) EP treatment (right).

[0020] [Figure 3B] Figure 1 shows a compound screen including untreated (-) and DMSO-treated (DMSO) controls under cold shock conditions [C] showing the effect on DNA repair outcome frequency. Data are presented as the mean ± SD of three biological replicates.

[0021] [Figure 3C] FIG. 3B shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 3B.

[0022] [Figure 3D] NU7441 and SCR7 combination treatment under cold shock conditions [C] and the effect on repair outcome frequency. Data are presented as the mean of two biological replicates, except for N+S, which is presented as the mean of four biological replicates ± SD.

[0023] [Figure 3E] FIG. 3D shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 3D.

[0024] [Figure 3F] Figure 1 shows combined treatment of cell cycle inhibitor XL413 (XL) after EP (24 h) with N+S before (4 h) and after EP (48 h) under standard culture conditions [A] or cold shock conditions [C]. Data are presented as mean ± SD of three technical replicates for each treatment.

[0025] [Figure 3G] Figure 1 shows the ratio of HDR / MutEJ repair outcomes in heterozygous GFP iPS cells treated with XL413 (XL) 24 hours after EP and NU7441+SCR7 (N+S) 4 hours before and 48 hours after EP under standard culture conditions [A] or cold shock conditions [C] 48 hours after EP. Statistical significance was calculated by unpaired two-tailed t-test, and P values ​​are indicated as **p<0.01, ***p<0.001. Single and combined treatments significantly improved HDR / MutEJ ratios compared to untreated controls (-).

[0026] [Figure 3H] Figure 13. Statistical analysis of single vs. combined treatments to address synergistic editing. Statistical significance was calculated by unpaired two-tailed t-test, with P values ​​indicated as *p<0.05, **p<0.01, ***p<0.001. For NEPA21 electroporation, XL+N+S treatment was significantly higher than XL alone, but not N+S alone. Combined cold-shock and N+S treatment was significantly higher compared to cold-shock treatment alone. Cold-shock with XL+N+S, combining all conditions, led to a significant improvement compared to cold-shock treatment alone or XL treatment alone, but not compared to cold-shock with N+S.

[0027] [Figure 3I] Figure 3G shows cell counts recorded for samples from day 8, when cells were collected for FACS analysis. Although a reduction in cell number was observed with each treatment, an additional reduction was observed in the combination treatment conditions, which may account for the high variability and the resulting low statistical significance. Possibly, the low viability resulting from the combination treatment with NEPA21 electroporation, especially the XL+N+S combination treatment, may have led to a higher replicative variability and therefore a lack of statistical significance. This may have occurred through clonal drift when cell numbers were low.

[0028] [Figure 3J] Representative FACS plots of cells treated with XL413 (XL) and N+S under standard conditions [A] (left) and cold shock conditions [C] (right) as tested in Figure 3F.

[0029] [Figure 4A] Figures 4A-G show homozygous fluorescent DNA repair assays to visualize and quantify allele-specific DNA repair outcomes during biallelic editing in iPS cells. Figure 4A is a schematic of the expected outcomes of GFP editing in homozygous AAVS1-CAG::EGFP (GFP) iPS cell lines and their distribution in FACS.

[0030] [Figure 4B] Figure 1 shows DNA repair outcome frequencies for single- versus biallelic editing in heterozygous and homozygous GFP iPS cells. HDR* indicates double positive BFP / GFP cells resulting from heterozygous editing and HDR repair in homozygous GFP iPS cells.

[0031] [Figure 4C] FIG. 4B shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 4B.

[0032] [Figure 4D] FIG. 4B shows quantification of biallelic repair outcomes in homozygous GFP iPS cells, derived from FACS gating on each possible DNA repair outcome presented in FIG. 4A.

[0033] [Figure 4E] FIG. 13 shows the effect of combined cold shock and drug treatment on repair outcome frequency in homozygous GFP iPS cells.

[0034] [Figure 4F] FIG. 4C shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 4E.

[0035] [Figure 4G] Figure 4B shows biallelic repair outcomes in the presence (N+S) or absence (-) of combined drug treatments, derived from the DNA repair outcomes presented in Figure 4E. All data are presented as the mean ± SD of three technical replicates for each treatment.

[0036] [Figure 5A] 5A-5E are diagrams showing that the creation of heterozygous mutations requires the protection of one allele. FIG. 5A shows the target sequence (SEQ ID NO: 65 and 66) in the GFP reporter and the ssODN repair template that creates a missense Y66H mutation (ssODN M, SEQ ID NO: 68 and 69), a silent T65T blocking mutation (ssODN B, SEQ ID NO: 70 and 71), or no mutation (ssODN W, SEQ ID NO: 72 and 73). ssODN B T65T becomes the protected GFP (pGFP) allele by HDR repair. Cells with unmodified or pGFP alleles are indistinguishable by FACS and are cumulatively scored as GFP.

[0037] [Figure 5B] Figure 1 shows GFP editing with ssODN M, ssODN B, or ssODN W alone, or with M+B and M+W combinations, and the effect on DNA repair outcome frequency under cold shock conditions. HDR* indicates the frequency of heterozygous double positive BFP / GFP repair outcomes or the frequency of compound heterozygous double positive BFP / pGFP repair outcomes. GFP positive cells include unmodified cells and HDR-mediated single allele pGFP / GFP repair outcomes, pGFP / insertion deletion (△) repair outcomes, or biallelic pGFP / pGFP repair outcomes.

[0038] [Figure 5C] Distribution of biallelic repair outcomes shown in Figure 5B.

[0039] [Figure 5D] FIG. 5C shows the biallelic BFP / GFP repair outcome frequency measured in FIG. 5C.

[0040] [Figure 5E] Representative FACS plots of BFP conversion only (ssODN M) or compound heterozygous BFP / pGFP mutations (ssODN M+B). All data are presented as mean ± SD of three technical replicates of independent electroporations for each condition.

[0041] [Figure 6A] Figures 6A-6H show synergistic gene editing at endogenous loci. Figure 6A shows DNA repair outcomes of heterozygous (Hetero) GFP iPS cells generated in 409B2 genetic background. Cells were targeted with Y66H mutant ssODN M alone (ssODN M) under standard culture conditions (37°C) or cold shock (32°C, 48 hours after EP), and treated alone or in combination with cell cycle inhibitor XL413 (XL) after EP (24 hours) and N+S before EP (4 hours) and after EP (48 hours).

[0042] [Figure 6B] Figure 3 shows the ratio of HDR / MutEJ repair outcomes in heterozygous GFP iPS cells using MaxCyte electroporation under the same treatment conditions as in Figure 3G. Statistical significance was calculated by unpaired two-tailed t-test, with P values ​​indicated as *p<0.05, **p<0.01, and ***p<0.001. With MaxCyte electroporation, which consistently showed higher cell viability, both the XL+N+S synergistic condition and the cold-shock and N+S synergistic condition showed significantly higher HDR / MutEJ ratios compared to the single treatments, supporting the finding that there is a synergistic effect when simultaneously regulating cell cycle and DNA repair.

[0043] [Figure 6C]Figure 1 shows DNA repair outcomes and HDR / MutEJ ratios of homozygous (Homo) GFP iPS cells generated in the 409B2 genetic background. Cells were targeted with Y66H mutant ssODN M only (ssODN M), processed and quantified as described in a. HDR* indicates the frequency of heterozygous double positive BFP / GFP.

[0044] [Figure 6D] FIG. 6C shows the ratio of HDR / MutEJ repair outcomes measured in FIG.

[0045] [Figure 6E] Figure 1 shows DNA repair outcomes and HDR / MutEJ ratios of homozygous (Homo) GFP iPS cells targeted with a combination of Y66H mutant ssODN M and T65T silent blocking ssODN B (ssODN M+B), treated and quantified as described in a. Here, HDR* indicates compound heterozygous double positive BFP / pGFP repair outcomes. GFP positive cells consist of unmodified cells and HDR-mediated monoallelic pGFP / GFP repair outcomes, pGFP / insert deletion (△) repair outcomes, or biallelic pGFP / pGFP repair outcomes.

[0046] [Figure 6F] Figure 6B shows the ratio of HDR / MutEJ repair outcomes measured in Figure 6E. Data are presented as the mean ± SD of three technical replicates for each treatment.

[0047] [Figure 6G]Figure 1 shows DNA repair outcome frequencies in 409B2 cells of single clones (n=32 or 31 as indicated) cultured under standard conditions (-), synergistic cell cycle inhibitors (XL+N+S) of XL413 after EP (24 hours) and NU7441+SCR7 before EP (4 hours) and after EP (48 hours), or synergistic cell cycle inhibitors (CS+N+S) of cold shock (32°C, 48 hours after EP). HDR outcomes indicate clones that have undergone template-mediated modification at one or both alleles. In other words, MutEJ outcomes include all clones with mutagenic indels at either allele, and wild-type outcomes correspond to clones with both unmodified alleles. Each targeting is labeled with the target gene and the desired missense mutation.

[0048] [Figure 6H] FIG. 6B shows the HDR / MutEJ ratios quantified in FIG. 6G for the same number of single clones (n=32 or 31 as indicated).

[0049] [Figure 7] Figure 1. Overview of synergistic gene editing effects relevant to HDR outcomes. Synchronization and release of cells into HDR-permissive cell cycle S and G2 phases synergizes with DNA repair inhibitors to improve HDR rates. Additional cell cycle-independent effects of cold shock may also play a role. Exploiting these mechanisms, homozygous mutations are created with high efficiency, while heterozygous mutations can be created without indels by using combinations of mutants and blocked ssODN templates.

[0050] [Figure 8A]Figures 8A-C show GFP-targeted clone production. Figure 8A is a schematic of the AAVS1 locus before (top) and after (bottom) targeting with a CAG-GFP cassette. Expected fragment sizes from SphI digestion are indicated, as well as genomic or endogenous Southern blot probe positions (5' or GFP probe). Grey boxes correspond to the PPP1R12C exon. HA-L / R: left / right homology arms, SA: splice acceptor, T2A: Thosea asigna virus 2A peptide, pA: bovine growth hormone or rabbit b-globin polyadenylation signals.

[0051] [Figure 8B] Southern blot analysis of SphI-digested genomic DNA with a 5' genomic probe (left) or an internal GFP probe (right). Targeted alleles were detected by the presence of a 3.5 kbp (5' probe) or 6.9 kbp (GFP probe) product, while the normal allele retaining the 6.5 kbp product was detected with the 5' probe. Clones with aberrant banding due to donor backbone integration, expected and observed at 8.5 kbp (5' probe) and 7.9 kbp (GFP probe), are indicated with an asterisk (*). Heterozygous 073-1-3 (#3) clone and homozygous 073-1-2 (#2) clone were selected for further gene editing experiments. Southern blots represent one incremental exposure from one membrane.

[0052] [Figure 8C]AAVS1 genotype (left), fluorescent microscopy images in bright field (center), and GFP fluorescence intensity by FACS (right) of the non-targeted 1383D6 parental iPS cell line (top), a GFP-targeted heterozygous cell line (073-1-3, middle), and a GFP-targeted homozygous cell line (073-1-2, bottom). Scale bar is 500 μm. Representative images from more than three independent experiments with similar results are shown.

[0053] [Figure 9A] Figures 9A-C show characterization of fluorescent DNA repair assays. Figure 9A shows targeting of heterozygous GFP iPS cells with plasmids expressing Cas9 and GFPx199 and targeting of heterozygous GFP iPS cells with ssODN (top center), selection with puromycin for 48 hours, and FACS sorting of HDR DNA repair outcomes (BFP+, bottom left), MutEJ DNA repair outcomes (double negative, bottom center), and unmodified DNA repair outcomes (GFP+, bottom right). FACS in the lower panel was performed 1 week after sorting the expanded cells. Control shows non-targeted heterozygous GFP iPS cells (top left).

[0054] [Figure 9B] Figure 9A shows the fragment length polymorphism (RFLP) assay of the NcoI restriction enzyme site introduced by ssODN-mediated targeting for the non-targeted (NT) control outcome, the heterozygous GFP (Hetero) control outcome, and the outcomes of HDR classification, MutEJ classification, and unmodified DNA classification obtained in Figure 9A. The proportion of HDR outcomes is shown by cleavage product quantification. RFLP analysis was performed once.

[0055] [Figure 9C] FIG. 1 shows a schematic (top left) and sequence (top right) of sorted heterozygous MutEJ cells, as well as TIDE analysis showing the resulting insertion-deletion patterns (bottom).

[0056] [Figure 10A] Figures 10A-10H show the effect of cell cycle synchronization on HDR efficiency. Figure 10A shows the experimental timeline (top) for no cold shock [A], cold shock 24 hours after electroporation (EP) [B] or cold shock 48 hours after electroporation (EP) [C], or cold shock 24 hours before EP only [D], or cold shock 24 hours before EP and cold shock 24 hours after EP [E] or cold shock 24 hours before EP and cold shock 48 hours after EP [F]. The effect obtained on DNA repair outcome frequency is shown for each condition [D-F] (bottom).

[0057] [Figure 10B] FIG. 10B shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 10A.

[0058] [Figure 10C] Figure 1 shows that cell cycle synchronization with nocodazole at G2 / M does not affect HDR efficiency in iPS cells. Figure 2 shows experimental timeline of nocodazole treatment for 16 h before EP under standard culture conditions [A] or under cold shock conditions after EP on day 0 [C] (top). EdU / PI staining was performed before EP and FACS analysis was performed on day 8. Figure 3 shows the resulting effect on DNA repair outcome frequency (bottom).

[0059] [Figure 10D] FIG. 10C shows the ratio of HDR / MutEJ repair outcomes measured in FIG.

[0060] [Figure 10E]Representative FACS plots of pre-EP EdU / PI staining of untreated and nocodazole-treated cells (top), cell cycle quantification (middle left), and mean EdU intensity of S-phase cells (middle right). Schematic of nocodazole-induced late G2 / M cell cycle arrest (bottom).

[0061] [Figure 10F] Figure 1 shows that cell cycle synchronization at the G1 / S boundary with XL413 improves HDR efficiency in iPS cells. Figure 2 shows the experimental timeline of XL413 treatment 24 hours before (Pre) or after (Post) EP under standard culture conditions [A] or cold shock conditions [C] (top). Figure 3 shows the resulting effect on DNA repair outcome frequency (bottom).

[0062] [Figure 10G] FIG. 10C shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 10F.

[0063] [Figure 10H] Relative cell numbers compared to untreated controls from Figure 10F. Pre- and post-XL413 treatment resulted in cell death and non-detectable (nd) repair outcomes in Figure 10F and Figure 10G. All data are presented as mean ± SD of three technical replicates for each treatment.

[0064] [Figure 11A] Figures 11A-11F show compound heterozygous mutations prevent Cafs9 re-cleavage. Figure 11A shows the generation of BFP / pGFP double positive clones using Y66H mutant ssODN M (SEQ ID NO: 77 and 78) and T65T silent blocking ssODN B (SEQ ID NO: 79 and 80) in homozygous GFP iPS cells. Double positive BFP / GFP cells were sorted on day 8 post-targeting and clones were picked and analyzed.

[0065] [Figure 11B] Figure 1 shows an experimental timeline of GFPx199 gRNA-targeted BFP / pGFP clones to measure Cas9-mediated re-cleavage activity of edited and protected GFP alleles under standard culture conditions, cold shock conditions, and NU7441+SCR7 combined (N+S) treatment (top). Figure 2 shows FACS plots of non-targeted BFP / pGFP clones (control) and re-targeted BFP / pGFP clones under standard culture [A], cold shock [C], and cold shock combined with N+S treatment (32°C+N+S) during FACS analysis on day 8 (bottom).

[0066] [Figure 11C] Figure 1 shows the effect of GFP editing with ssODN M, ssODN B, or ssODN W alone, or M+B and M+W combinations, and the frequency of DNA repair outcomes under cold shock and N+S treatment. HDR* indicates the frequency of heterozygous double positive BFP / GFP repair outcomes or the frequency of compound heterozygous double positive BFP / pGFP repair outcomes. GFP positive cells include unmodified cells and HDR-mediated single allele pGFP / GFP repair outcomes, pGFP / insertion deletion (△) repair outcomes, or biallelic pGFP / pGFP repair outcomes.

[0067] [Figure 11D] Distribution of biallelic repair outcomes shown in FIG. 11C.

[0068] [Figure 11E] FIG. 11D shows the frequency of biallelic BFP / GFP repair outcomes measured in FIG.

[0069] [Figure 11F]Representative FACS plots of BFP conversion only (ssODN M) or compound heterozygous BFP / pGFP mutations (ssODN M+B). Data are presented as the mean ± SD of three technical replicates of independent electroporations for each condition.

[0070] [Figure 12A] 12A-12F are diagrams showing the creation of heterozygous mutations at the endogenous locus. FIG. 12A shows the target sequence and editing strategy at the ATP1A1 locus to generate the p.Q118R and p.N129D mutations. Sanger sequences of the ATP1A1 locus before and after HDR editing are shown.

[0071] [Figure 12B] Experimental timeline for ATP1A1 editing including cold-shock selection, combined treatment selection, and ouabain selection (top). Quantification of HDR colony formation using a combination of cold-shock [C] and N+S drug treatment (bottom). Data are presented as mean ± SD of four technical replicates for each treatment.

[0072] [Figure 12C] 1 shows the target sequence of the KCNH2 locus and the strategy to reconstitute the heterozygous p.N588K pathogenic mutation with a ssODN encoding a missense mutation (ssODN M) or a silent interruption (ssODN B) using a combination of cold shock and N+S treatment. Sanger sequences of the KCNH2 locus before and after HDR editing with mixed ssODN repair templates are shown.

[0073] [Figure 12D] Figure 1 shows the target sequence of the PSMB8 locus and the strategy to reconstitute the heterozygous p.G201V pathogenic mutation. Also shown are the Sanger sequences of the PSMB8 locus before and after HDR editing with mixed ssODN repair templates.

[0074] [Figure 12E] FIG. 13 shows quantification of editing efficiency of compound heterozygous, homozygous, and heterozygous mutations obtained from clonal analysis of editing with ssODN M (n=96) or ssODN M+B (n=92).

[0075] [Figure 12F] FIG. 12C shows that ssODN design and quantification is similar to FIG. 12E when ssODN M (n=91) or ssODN M+B (n=95) are used.

[0076] [Figure 13A] Figures 13A-H show the increase in editing efficiency using MaxCyte electroporation. Figure 13A shows the frequency of DNA repair outcomes using the MaxCyte electroporation device. Heterozygous (Hetero) GFP iPS cells and homozygous (Homo) GFP iPS cells produced in a 1383D6 genetic background were targeted with Y66H mutant ssODN M alone (ssODN M) and homozygous GFP iPS cells were targeted with a combination of Y66H mutant ssODN M and T65T silently blocked ssODN B (ssODN M+B) under standard culture conditions (37°C), cold shock conditions (32°C, 48 hours after EP), or cold shock conditions combined with N+S treatment. HDR* indicates the frequency of heterozygous double positive BFP / GFP repair outcomes and the frequency of compound heterozygous double positive BFP / pGFP repair outcomes. For homozygous GFP iPS cells targeted with ssODN M+B, GFP positive cells include unmodified cell repair outcomes, and HDR-mediated monoallelic pGFP / GFP repair outcomes, HDR-mediated monoallelic pGFP / indel (△) repair outcomes, or HDR-mediated biallelic pGFP / pGFP repair outcomes.

[0077] [Figure 13B] FIG. 13B shows the ratio of HDR / MutEJ repair outcomes measured in FIG. 13A.

[0078] [Figure 13C] FIG. 13B shows the results of an experiment similar to that of FIG. 13A, but with heterozygous and homozygous GFP iPS cells produced in the 409B2 genetic background.

[0079] [Figure 13D] FIG. 13B shows the results of an experiment similar to that of FIG. 13B, but with heterozygous and homozygous GFP iPS cells produced in the 409B2 genetic background.

[0080] [Figure 13E] FIG. 13B shows the frequency of biallelic repair outcomes in homozygous GFP iPS cells in the 1383D6 genetic background targeted with ssODN M under standard culture conditions, cold shock conditions, or cold shock conditions combined with N+S treatment, taken from FIG. 13A.

[0081] [Figure 13F] This figure shows the results of an experiment similar to that of FIG. 13E of homozygous GFP iPS cells in a 1383D6 genetic background targeted with ssODN M+B under standard culture conditions, cold shock conditions, or cold shock conditions combined with N+S treatment, and the effects on biallelic BFP / BFP efficiency and on compound heterozygous BFP / pGFP efficiency.

[0082] [Figure 13G] FIG. 13C shows the results of an experiment similar to that of FIG. 13E, but using homozygous GFP iPS cells generated in a 409B2 genetic background. All data are presented as the mean ± SD of three technical replicates for each treatment.

[0083] [Figure 13H] FIG. 13C shows the results of an experiment similar to that of FIG. 13F, but using homozygous GFP iPS cells generated in a 409B2 genetic background. All data are presented as the mean ± SD of three technical replicates for each treatment.

[0084] [Figure 14A] Figures 14A-D show synergistic gene editing using MaxCyte electroporation. Figure 14A shows the frequency of biallelic repair outcomes obtained from homozygous GFP iPS cells targeted with ssODN M during single or combined treatment with cell cycle inhibitors XL413 (XL) and N+S under standard culture conditions.

[0085] [Figure 14B] FIG. 14B shows the results of an experiment similar to that of FIG. 14A, but under cold-shock conditions.

[0086] [Figure 14C] FIG. 1 shows the frequency of biallelic repair outcomes obtained from homozygous GFP iPS cells targeted with ssODN M+B under standard culture conditions.

[0087] [Figure 14D] FIG. 14C shows the results of an experiment similar to that in C, but under cold-shock conditions. All data are presented as the mean ± SD of three technical replicates for each treatment.

[0088] [Figure 15A] Figure 15A-J shows the sequence design of the endogenous locus targeted using synergistic gene editing and MaxCyte electroporation. The target sequences and editing strategies for all 10 targeting experiments, including gRNA and ssODN template sequences, and missense mutations that have been created. Figure 15A shows the sequence design for KCNH1 D85N gene editing.

[0089] [Figure 15B] FIG. 1 shows the sequence design for KCNH2 N45D gene editing.

[0090] [Figure 15C]FIG. 1 shows the sequence design for SCN5A A1428S gene editing.

[0091] [Figure 15D] FIG. 1 shows the sequence design for KCNH2 N588D gene editing.

[0092] [Figure 15E] FIG. 1 shows the sequence design for KCNH2 N588K gene editing.

[0093] [Figure 15F] FIG. 1 shows the sequence design for APRT M136T gene editing.

[0094] [Figure 15G] FIG. 1 shows the sequence design for HES7 R25W gene editing.

[0095] [Figure 15H] FIG. 1 shows the sequence design for PSMB8 G201V gene editing.

[0096] [Figure 15I] FIG. 1 shows the sequence design for KCNJ11 T293N gene editing.

[0097] [Figure 15J] FIG. 1 shows the sequence design for KCNJ11 T294M gene editing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] [Method for producing genome-edited cells] In one embodiment, the invention provides a method for producing a genome-edited cell, comprising: synchronizing a cell cycle of the cell; treating the cell with an agent that alters a DNA repair pathway; introducing donor DNA into the cell; and inducing a sequence-specific double-stranded break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a portion of the base sequence of the donor DNA, thereby obtaining the cell with an edited genome.

[0099] As described below in the Examples, the inventors of the present application have demonstrated that the manufacturing method of this embodiment can improve the efficiency of the homology-directed repair pathway or an equivalent pathway, and can perform precise genome editing.

[0100] The target cells for genome editing may be human cells or non-human animal cells. Non-human animals are not particularly limited, and examples thereof include mice, rats, rabbits, pigs, sheep, goats, cows, monkeys, etc.

[0101] The cells may be somatic cells or stem cells. Examples of stem cells include pluripotent stem cells and tissue stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include hematopoietic stem cells, mesenchymal stem cells, neural stem cells, muscle stem cells (satellite cells), skin stem cells, and the like. The cells may be cultured cells, cells constituting an animal embryo, or cells constituting a living organism.

[0102] In the manufacturing method of this embodiment, the donor DNA may be any DNA, linear or circular, double-stranded or single-stranded, and may be isolated, synthesized, or enzymatically produced. More specifically, a PCR fragment, a plasmid, a single-stranded oligonucleotide, or a double-stranded oligonucleotide may be used as the donor DNA. Among these, a single-stranded donor oligonucleotide (ssODN) is preferred. The length of the single-stranded donor oligonucleotide may be about tens to hundreds of bases, and may be about 100 bases.

[0103] The step of inducing a sequence-specific double-strand break in the genomic DNA of the cell is preferably carried out by introducing into the cell a sequence-specific DNA cleaving enzyme, as described below.

[0104] The donor DNA and the sequence-specific DNA cleaving enzyme may be introduced into the cells using a gene transfer reagent or electroporation.

[0105] Examples of gene transfer reagents that can be used include Lipofectamine 2000, Lipofectamine 3000, CRISPRMAX and RNAMAX (all from Thermo Fisher Scientific), FuGENE 6 and FuGENE HD (all from Promega), etc.

[0106] Electroporation can be performed using equipment such as NEPA21 (Nepgene Co., Ltd.), MaxCyte STX (MaxCyte), Neon (Thermo Fisher Scientific), and 4D-Nucleofector (Lonza).

[0107] Preferably, the step of synchronizing the cell cycle of the cells includes incubating the cells at 25° C. to 35° C., preferably about 32° C., for 24 hours to 72 hours, preferably about 48 hours. (Hereinafter, this process may be referred to as "cold shock").

[0108] The cold shock is preferably performed immediately after the introduction of the donor DNA and the sequence-specific DNA cleaving enzyme into the cells. The cold shock not only synchronizes the cell cycle but also has the effect of improving the efficiency of the homology-directed repair pathway or an equivalent pathway through other unknown effects.

[0109] The step of synchronizing the cell cycle of the cells preferably includes treating the cells with a Cell Division Cycle 7 (CDC7) inhibitor. Examples of CDC7 inhibitors include XL413 (CAS number: 1169558-38-6).

[0110] XL413 is preferably added to the cell culture medium at a final concentration of 1 to 100 μM, preferably about 33 μM. XL413 is preferably added to the medium immediately after introduction of the donor DNA and the sequence-specific DNA cleaving enzyme into the cells. XL413 is preferably incubated with the cells for about 24 hours.

[0111] More preferably, the step of synchronizing the cell cycle of the cells is carried out by utilizing cold shock in combination with a CDC7 inhibitor.

[0112] Preferably, the agent that modifies the DNA repair pathway includes a DNA-PK inhibitor or a DNA ligase IV inhibitor. More preferably, the agent that modifies the DNA repair pathway includes a combination of a DNA-PK inhibitor and a DNA ligase IV inhibitor.

[0113] An example of a DNA-PK inhibitor includes NU7441 (CAS number: 503468-95-9). NU7441 is preferably added to the cell culture medium at a final concentration of 100 nM to 10 μM, preferably about 2 μM.

[0114] An example of a DNA ligase IV inhibitor includes SCR7 (CAS number: 14892-97-8). SCR7 is preferably added to the cell culture medium at a final concentration of 100 nM to 10 μM, preferably about 1 μM.

[0115] Preferably, the agent that alters a DNA repair pathway is incubated with the cells from about 4 hours prior to and about 48 hours after introduction of the donor DNA and the sequence-specific DNA cleaving enzyme into the cells.

[0116] Generally, the sequence-specific DNA cleavage enzyme used to induce DSB for genome editing is roughly classified into RNA-guided nuclease and artificial nuclease. The sequence-specific DNA cleavage enzyme may be an RNA nuclease or an artificial nuclease.

[0117] Sequence-specific DNA cleavage enzymes cleave genomic DNA in a target sequence-specific manner to create double-strand breaks. There is no particular limitation as long as it forms a target sequence. The length of the target sequence recognized by the sequence-specific DNA cleaving enzyme can be, for example, about 10 to 60 bases.

[0118] In addition, the sequence-specific DNA cleavage enzyme may be, for example, in the form of multiple nickases binding to cleave DNA. Nickase refers to an enzyme that forms a nick in a single strand of double-stranded DNA. For example, a double-stranded break can be formed by forming nicks in both strands of double-stranded DNA at adjacent positions on genomic DNA.

[0119] RNA-guided nucleases are enzymes that bind to a target sequence using a short guide RNA and recruit a nuclease with two DNA cleavage domains (nuclease domains) to induce sequence-specific cleavage. Examples of RNA-guided nucleases include CRISPR-Cas family proteins.

[0120] Examples of CRISPR-Cas family proteins include Cas9, Cas12 (Cpf1), Cas13 (C2c2), CasX, CasY, etc. The RNA-guided nuclease may be a homolog of a CRISPR-Cas family protein or a modified CRISPR-Cas family protein. For example, it may be a nickase-modified nuclease in which one of the two wild-type nuclease domains is modified to be inactive. Alternatively, it may be Cas9-HF, HiFi-Cas9, eCas9, etc., which have improved target specificity.

[0121] Examples of Cas9 include those derived from Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus, etc. Examples of Cpf1 include those derived from Acidaminococcus, Krasnopyra, Chlamydomonas, Francisella novicida, etc.

[0122] An artificial nuclease is an artificial restriction enzyme that has a DNA binding domain designed and engineered to specifically bind to a target sequence and a nuclease domain (such as the DNA cleavage domain of the restriction enzyme FokI). Examples of artificial nucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, etc.

[0123] In the production method of this embodiment, the sequence-specific DNA cleaving enzyme is preferably a ribonucleoprotein (RNP) complex containing Cas9 and gRNA.

[0124] When CRISPR-Cas is used as the sequence-specific DNA cleavage enzyme introduced into cells, the target base sequence is determined by gRNA. gRNA may be introduced into cells in the form of RNA, or may be introduced into cells in the form of expression vector and expressed in cells.

[0125] Examples of methods for preparing gRNA in the form of RNA include a method in which a construct is prepared in which a promoter such as T7 is added upstream of a nucleic acid fragment encoding gRNA, and then synthesized by an in vitro transcription reaction, a method in which gRNA is chemically synthesized, etc. When gRNA is chemically synthesized, chemically modified RNA may be used.

[0126] Examples of expression vectors include plasmid vectors or viral vectors that transcribe the gRNA from a Pol III promoter, such as the H1 promoter or the U6 promoter. When the gRNA is expressed using an expression vector, the gRNA may be expressed constitutively or under the control of an inducible promoter.

[0127] The site-specific double-stranded cleavage site is determined by the approximately 20-base protospacer sequence of the gRNA and an approximately 3- to 5-base protospacer adjacent motif (PAM) that binds to the target sequence in a complementary manner.

[0128] The gRNA may be a complex of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), or a single synthetic gRNA (sgRNA) that is a combination of tracrRNA and crRNA.

[0129] Induction of a sequence-specific double-strand break in the genomic DNA of a cell in the presence of donor DNA results in repair of the double-strand break by a homology-directed repair pathway or an equivalent pathway following all or part of the donor DNA sequence, or the sequence-specific double-strand break is repaired by a pathway involving insertion or deletion mutations. Pathways equivalent to homology-directed repair may include single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA), among others.

[0130] In the production method of this embodiment, the donor DNA may have a nucleotide sequence that modifies the protospacer sequence or the protospacer adjacent motif (PAM) sequence.

[0131] When the double-stranded break formed by the RNP complex containing Cas9 and gRNA is repaired by the homologous recombination repair pathway or an equivalent pathway according to all or part of the nucleotide sequence of the donor DNA having a mutation that alters the protospacer sequence or the protospacer adjacent motif (PAM) sequence, it is possible to prevent the allele from being re-cut by the RNP complex, i.e., it is possible to prevent the target site from being cut again by the RNP complex and introducing an insertion or deletion mutation during re-repair.

[0132] In the manufacturing method of this embodiment, the donor DNA may have a mutation, and a cell may be obtained in which the mutation is introduced into one or both alleles. The mutation may be a naturally occurring mutation or a designed mutation. A naturally occurring mutation is a mutation corresponding to a gene polymorphism or variant, or in other words, an existing variant having benign, pathogenic, or unknown function detected in the human population. A designed mutation is an artificially designed mutation.

[0133] The donor DNA may include a combination of multiple donor DNAs corresponding to the same region on the genome. For example, the donor DNA includes a combination of a first donor DNA and a second donor DNA, where the first donor DNA has a mutation of interest and the second donor DNA has a mutation that modifies the protospacer sequence or the PAM sequence, and the mutation of interest is introduced into only one allele, and a cell can be obtained in which the protospacer sequence or the PAM sequence of the other allele is modified.

[0134] The first donor DNA has a mutation of interest. The mutation of interest is the mutation of interest, or in other words, the mutation that is intended to be introduced into the cell. Examples of mutations of interest include mutations that revert disease-causing mutations to wild type.

[0135] When the sequence-specific double-stranded break is repaired by a homology directed repair pathway or an equivalent pathway according to all or part of the nucleic acid sequence of the second donor DNA, the allele is protected from re-cleavage by the RNP complex.

[0136] When the donor DNA comprises a combination of first and second donor DNA, the first donor DNA introduces the mutation of interest into one allele, while the other allele is protected by the second donor DNA, facilitating the production of heterozygous mutants.

[0137] [Methods for editing the genome of cells] In one embodiment, the invention provides a method of editing the genome of a cell, comprising synchronizing a cell cycle of the cell, treating the cell with an agent that alters a DNA repair pathway, introducing donor DNA into the cell, and inducing a sequence-specific double-stranded break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by homologous recombination or an equivalent pathway based on all or a portion of the base sequence of the donor DNA, resulting in the cell having an edited genome.

[0138] As described below in the Examples, the inventors of the present application have demonstrated that the manufacturing method of this embodiment can improve the efficiency of the homology-directed repair pathway or an equivalent pathway, and can perform precise genome editing.

[0139] In the method of this embodiment, the cells to be subjected to genome editing, the donor DNA, the step of synchronizing the cell cycle of the cells, the agent that modifies the DNA repair pathway, the step of inducing sequence-specific double-stranded breaks in the genomic DNA of the cells in the presence of the donor DNA, etc. are the same as those described above.

[0140] [Methods for detecting gene editing on one or both alleles] In one embodiment, the present invention provides a method for detecting gene editing on one or both alleles, the method comprising: introducing donor DNA into a cell, the cell having a gene encoding a fluorescent protein that emits a first fluorescence in both alleles, the donor DNA having a mutation that changes the first fluorescence of the fluorescent protein to a second fluorescence; inducing a sequence-specific double-stranded break in genomic DNA of the cell in the presence of the donor DNA, such that the double-stranded break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA to obtain a genome-edited cell; and analyzing the fluorescence of the genome-edited cell, whereby, if (i) a first fluorescence is detected and a second fluorescence is not detected, (i-1) both alleles of the fluorescent protein locus have not changed, or (i-2) one allele of the fluorescent protein locus has not changed and an insertion or deletion mutation has occurred in the other allele. (ii) if the first fluorescence is not detected and the second fluorescence is detected, (ii-1) it is indicated that both alleles of the fluorescent protein locus have been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the nucleotide sequence of the donor DNA, or (ii-2) it is indicated that one allele of the fluorescent protein locus has been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the nucleotide sequence of the donor DNA, and an insertion mutation or deletion mutation has occurred in the other allele; (iii) if neither the first fluorescence nor the second fluorescence is detected, it is indicated that an insertion mutation or deletion mutation has occurred in both alleles of the fluorescent protein locus; and (iv) if both the first fluorescence and the second fluorescence are detected, it is indicated that one allele of the fluorescent protein locus has been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the nucleotide sequence of the donor DNA, and the other allele has not changed.

[0141] According to the method of this embodiment, it becomes easy to visualize and quantitatively analyze whether DNA repair in genome editing targeting one or both alleles was carried out by the homologous recombination repair pathway or an equivalent pathway, or whether an insertion or deletion occurred.

[0142] For example, by carrying out the method of this embodiment in the presence of a test substance, it is possible to screen for a substance that increases or decreases the efficiency of homology-directed repair.

[0143] In the method of this embodiment, the fluorescent protein that emits the first fluorescence may be a green fluorescent protein (GFP), the fluorescent protein that emits the second fluorescence may be a blue fluorescent protein (BFP), and the donor DNA may have a mutation that replaces the 66th tyrosine residue of GFP with a histidine residue to modify it into BFP.

[0144] In this case, by detecting whether the fluorescence emitted by the cells is GFP fluorescence or BFP fluorescence, it is possible to visualize and quantitatively analyze what type of DNA repair has been carried out.

[0145] Alternatively, the fluorescent protein that emits a first fluorescence may be GFP, the fluorescent protein that emits a second fluorescence may be cyan fluorescent protein (CFP), and the donor DNA may have a mutation that replaces the 66th tyrosine residue of GFP with a tryptophan residue to modify it into CFP.

[0146] In this case, by detecting whether the fluorescence emitted by the cells is GFP fluorescence or CFP fluorescence, it is possible to visualize and quantitatively analyze what type of DNA repair has been carried out.

[0147] [Genome editing kit] In one embodiment, the present invention provides a kit for genome editing comprising a CDC7 inhibitor, a DNA-PK inhibitor, and a DNA ligase IV inhibitor.

[0148] The kit of this embodiment can be suitably used for the method for producing a genome-edited cell and the method for editing the genome of a cell, as described above. By using the kit of this embodiment, the efficiency of the homologous recombination repair pathway or an equivalent pathway can be improved, and precise genome editing can be performed.

[0149] In the kit of this embodiment, the CDC7 inhibitor, DNA-PK inhibitor, and DNA ligase IV inhibitor are the same as those described above.

[0150] The kit of this embodiment may further include a sequence-specific DNA cleavage enzyme and a donor DNA. The sequence-specific DNA cleavage enzyme and the donor DNA are the same as those described above.

[0151] In the kit of this embodiment, the donor DNA may have a mutation that replaces the tyrosine residue at position 66 of GFP with a histidine residue to modify it into BFP.

[0152] In this case, by carrying out the above-described method for detecting genome editing in one or both alleles of a cell having a gene encoding GFP in both alleles, it becomes easy to visualize and quantitatively analyze DNA repair in genome editing targeting one or both alleles, regardless of whether it is carried out by the homologous recombination repair pathway or an equivalent pathway, or whether an insertion or deletion has occurred. EXAMPLES

[0153] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0154] [method] (Human iPS cell culture) Human induced pluripotent stem (iPS) cell lines, namely 409B2 (RIKEN BioResource Research Center (RIKENBRC) #HPS0076) and 1383D6 (RIKEN BioResource Research Center #HPS1006), were maintained in commercially available StemFit AK02N medium (Ajinomoto, Cat. No. RCAK02N) on cell culture plates coated with 0.5 mg / mL silk iMatrix-511, Recombinant Human Laminin-511 E8 Fragment (Nippi, Cat. No. 892021) at 37 °C, 5% CO2, and daily medium changes. Cell passage was performed every 7 days during maintenance. Cells were first detached with Accumax (Innovative Cell Technologies, Cat. No. AM105-500) and incubated at 37°C for 10 min, washed in StemFit AK02N medium supplemented with 10 μM ROCK inhibitor Y-27632 (Wako, Cat. No. 253-00513), and cultured at a density of 1 × 10 3 cells / cm 2 Cell lines were seeded at 100 ng / ml onto iMatrix-511-coated plates and cultured in StemFit AK02N medium supplemented with ROCK inhibitor for 48 h after seeding and without ROCK inhibitor thereafter. All cell lines were routinely tested negative for mycoplasma contamination.

[0155] (AAVS1 targeting) Cell lines heterozygously or homozygously targeted with a CAG-driven GFP cassette at the AAVS1 safe-harbor locus were established in two distinct human iPS cell genetic backgrounds, 409B2 and 1383D6. GFP heterozygous (317-A4) and GFP homozygous (317-D6) clones generated in the 409B2 parental iPS cell line were previously described43. Using a neomycin (E182) donor vector and TALE nuclease, GFP heterozygous (073-1-3) and GFP homozygous (073-1-2) clones were generated in the 1383D6 parental iPS cell line as previously described43. Genotyping was performed in targeted clones by Southern blotting using SphI digestion of genomic DNA, endogenous DIG-labeled probes or genomic DIG-labeled probes (GFP probe or 5' probe), and chemiluminescence detection for 2.5 h.

[0156] (iPS cell gene editing) Equimolar amounts of crRNA and tracrRNA sequences (IDT, Alt-R CRISPR-Cas9 crRNA and tracrRNA) were hybridized at 95°C for 5 min to form functional crRNA:tracrRNA duplexes (gRNA). For each electroporation (EP), 30.5 pmol of gRNA was mixed with 30.5 pmol of Cas9 nuclease (IDT, Alt-R SpCas9 Nuclease V3) (1:1 gRNA:Cas9 ratio) to form RNP complexes, which were then incubated at room temperature (RT) for 30 min. For compound heterozygous mutants, 82 pmol of ssODN repair template (IDT, Ultramer DNA Oligonucleotides) or an equal amount of mixed ssODN repair template totaling 82 pmol was added to the previously formed RNP complexes just before EP. The gRNA and ssODN sequences are listed in Tables 1 to 4, respectively. Cells were harvested with Accumax, washed, counted, and cultured in Opti-MEM I serum-free medium (Life Technologies, Cat. No. 31985-062) at a density of 5 × 10 5Resuspended at 5 x 10 cells / 10 μL 5 The cells were added to the mixture of RNP complex and ssODN, and a total volume of 25 μl of the suspension was electroporated in a Nepa Electroporation Cuvette 1 mm gap (Nepa Gene, Cat. No. EC-001) using a NEPA21 Electroporter (Nepa Gene) device (Pore pulse: 125 V voltage, pulse length 2.5 ms, pulse gap 50 ms, 2 pulse mode, 10% pulse decay, + orientation, Transfer pulse: 20 V voltage, pulse length 50 ms, pulse gap 50 ms, 5 pulse mode, 40% pulse decay, ± orientation). The electroporated cells were then transferred to iMatrix-511-coated plates in StemFit AK02N medium supplemented with ROCK inhibitor and incubated at 37°C or incubated at 37°C for 48 hours after incubation at 32°C for cold shock treatment. A medium change was performed with StemFit AK02N without ROCK inhibitor after 48 hours, and cells were maintained normally until FACS analysis on day 8. For editing endogenous loci in 409B2 iPS cells, 61 pmol of gRNA and 61 pmol of Cas9 were used to form an RNP complex (maintaining a 1:1 gRNA:Cas9 ratio), and an equal amount of 82 pmol of ssODN repair template was added to this RNP complex before EP, or 41 pmol of either mixed ssODN repair template was added if compound heterozygous mutants were to be produced. Then, 1 × 10 6 Cells were added to the mixture of RNP complexes and ssODN, and a total volume of 40 μl of the suspension was electroporated in a 1 mm gap Nepa Electroporation Cuvette using a NEPA21 electroporator under the same electroporation conditions outlined above.

[0157] [Table 1]

[0158] [Table 2] [Table 3] [Table 4]

[0159] In the case of plasmid-based delivery, target-specific gRNA sequences (GFPx199 gRNA: caccGCTGAAGCACTGCACGCCGT (SEQ ID NO:1) sense and aaacACGGCGTGCAGTGCTTCAGC (SEQ ID NO:2) antisense oligonucleotides) were cloned into pX459v2 vector (a gift from F. Zhang, Addgene #62988). 3 μg of targeting vector was then mixed with 3 μg (98 pmol) of ssODN repair template, and this mixture was supplemented with 1 × 10 6 Cells were added and a total of 100 μl of the mixed suspension was electroporated in a Nepa Electroporation Cuvette 2 mm gap (Nepa Gene, Cat. No. EC-002) using a NEPA21 electroporter (pore pulse: 125 V voltage, pulse length 5 ms, pulse gap 50 ms, 2 pulse mode, 10% pulse decay, + orientation, transfer pulse: 20 V voltage, pulse length 50 ms, pulse gap 50 ms, 5 pulse mode, 40% pulse decay, ± orientation). The electroporated cells were then transferred to iMatrix511-coated plates in StemFit AK02N medium supplemented with ROCK inhibitor and treated with 1 μg / ml puromycin (Merck, Cat. No. P7255-25MG) for 48 hours starting from 24 hours after EP. A medium change was then performed using StemFit AK02N without ROCK inhibitor and cells were maintained as normal until FACS analysis on day 8.

[0160] For gene editing experiments performed on a MaxCyte STX (MaxCyte) instrument, cells were first harvested 1 day before EP and grown at a density of 1.5 × 10 6Cells were plated on iMatrix-511-coated 6-well plates in StemFit AK02N medium supplemented with ROCK inhibitor. On the day of electroporation, RNP complexes were formed in separate 1.5 ml tubes by mixing 61 pmol of gRNA with 61 pmol of Cas9 nuclease (1:1 gRNA:Cas9 ratio) and 328 pmol of ssODN repair template. Cells were harvested, counted, and 2 × 10 6 The cells were resuspended in MaxCyte buffer (MaxCyte, Cat. No. EPB-1) at a density of 1 × 10 cells / 100 μL. 6 Cells were added to the RNP and ssODN mixture and a total volume of 50 μl was electroporated in the OC-100 (MaxCyte, Cat. No. SOC-1) or OC-100×2 (MaxCyte, Cat. No. SOC-1×2) processing assembly of MaxCyte STX (Optimized Protocol 8). After electroporation, the processing assembly was incubated at 37° C. for 20 min, and then the electroporated cells were transferred to iMatrix-511-coated plates in StemFit AK02N medium supplemented with ROCK inhibitor and incubated at 37° C. or at 32° C. for cold shock treatment. A medium change was performed after 48 h with StemFit AK02N without ROCK inhibitor and cells were maintained as normal until FACS analysis on day 8.

[0161] (Small molecule processing) For screening and optimization purposes, 5 μg of Cas9 (30.5 pmol) was set as the standard editing condition, although higher amounts of Cas9 resulted in higher editing efficiency. All small molecules were dissolved in dimethyl sulfoxide (DMSO) as recommended in the manufacturer's instructions, except for XL413, which was dissolved in water. The maximum non-toxic concentrations in iPS cells after 48 h of treatment, as defined by titration curves and colony calculations, were KU-55933 (Selleck Chemicals, Cat. No. S1092) 3 μM, VE-821 (Selleck Chemicals, Cat. No. S8007) 1 μM, NU7441 (Tocris Bioscience, Cat. No. 3712) 2 μM, Mirin (Sigma-Aldrich, Cat. No. M9948-5MG) 3 μM, PFM01 (Sigma-Aldrich, Cat. No. SML1735-5MG) 10 μM, TDRL-505 (Calbiochem, Cat. No. 530535) 20 μM, RS-1 (Sigma-Aldrich, Cat. No. R9782-5MG) 10 μM, and SCR7 (Xcess Small molecules were supplemented in StemFit AK02N medium at final concentrations of 1 μM (Sigma-Aldrich, Cat. No. M60082-2S), 1 μM (Sigma-Aldrich, Cat. No. M60082-2S), and 5 μM (Sigma-Aldrich, Cat. No. SML1362-5MG). XL413 (Sigma-Aldrich, Cat. No. SML1401-5MG) was supplemented at a final concentration of 33 μM, and nocodazole (Sigma-Aldrich, Cat. No. M1404-2MG) was supplemented at a final concentration of 100 ng / mL. When several small molecules were combined, concentrations were equivalent to single treatments. DMSO controls were supplemented with an amount equal to the maximum amount of small molecule alone or in combination used in the treatment condition. For gene editing, cells were pretreated with each small molecule for 4 h before EP and treated for 48 h after EP (pre), or, where indicated, were treated only 48 h after EP without pretreatment (post). Pretreated cells were harvested and resuspended in medium supplemented with small molecules during cell counting, and then resuspended in Opti-MEM medium for electroporation.After EP, cells were transferred to iMatrix-511-coated plates in StemFit AK02N medium supplemented with ROCK inhibitor and small molecules and incubated at 37°C or at 32°C for 48 hours followed by cold shock treatment and incubation at 37°C. A medium change was performed 48 hours after EP with StemFit AK02N medium and cells were maintained as standard until FACS analysis on day 8. For cell cycle synchronization and gene editing, XL413 treatment was performed 24 hours after EP and replaced with StemFit AK02N medium supplemented with ROCK inhibitor for 24 hours, after which it was replaced with StemFit AK02N medium without ROCK inhibitor. Nocodazole treatment was performed 16 hours before EP, supplemented with nocodazole during cell collection and counting, and replaced with StemFit AK02N medium supplemented with ROCK inhibitor after EP.

[0162] (cell cycle analysis) In addition to testing small molecules or culture conditions, cells were first incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h 15 min using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific, Cat. No. C10424). Cells were then harvested, counted, and an equal number of cells transferred to a new 1.5 ml tube. According to the manufacturer's instructions, cells were washed with 1% BSA in 1X DPBS, fixed with Click-iT fixative (4% paraformaldehyde in DPBS), incubated at room temperature (RT) and protected from light for 15 min, and washed with 1% BSA in DPBS. Cells were then permeabilized with 1X Click-iT saponin-based permeabilization and washing reagent. Click-iT labeling reaction was carried out by adding a mixture of Alexa Fluor 647 azide fluorescent dye, 2 mM CuSO4, and 1X Click-iT EdU buffer additive to the sample, incubating the reaction mixture at room temperature and protected from light for 30 min, and then washing and resuspending the sample with 1X Click-iT saponin permeabilization and washing reagent. Finally, propidium iodide (PI) (Wako, Cat. No. 169-26281) staining solution consisting of a final concentration of 0.1% Triton-X 100 (Nacalai Tesque, Cat. No. 282-29), 0.2 mg / mL RNase A (Invitrogen, Cat. No. 12091-021), and 20 μg / mL propidium iodide (PI) in DPBS was mixed with the sample and incubated at room temperature and protected from light for 30 min.

[0163] (Flow Cytometry and Cell Sorting) 5 × 10 cells resuspended in FACS buffer (2% FBS in DPBS). 5Fluorescence intensity measurements were performed on a BD LSRFortessa Cell Analyzer (BD Biosciences) using BD FACSDiva software (BD Biosciences) using 100 cells, and raw data were analyzed with FlowJo (FlowJo LLC). For editing experiments in GFP iPS cells, cells were acquired using Pacific Blue (450 / 50 nm) and FITC (530 / 30 nm) filters. Cells stained with EdU and PI were acquired using Alexa Fluor 647 (660 / 20 nm) and PE-Tx-Red-GR filters. For characterization of DNA repair outcomes, BFP+, BFP- / GFP-, and GFP+ groups were sorted from edited heterozygous GFP iPS cells, and for clonal isolation of compound heterozygotes, BFP+ / GFP+ groups were sorted from edited homozygous GFP iPS cells targeted with mixed ssODN repair templa / cates. Cells were cultured at a density of 1 × 10 6 Cells / ml were collected in FACS buffer and filtered through a cell strainer for separation. A sorting gate was set up for singlets, and the desired population was collected on a BD FACSAria II cell sorter (BD Biosciences) and placed in StemFit AK02N medium containing 10 μM–20 μM ROCK inhibitor.

[0164] (HDR quantification assay) Ouabain octahydrate (Sigma-Aldrich, Cat. No. O3125-250MG) was dissolved in water. To selectively obtain colonies edited by HDR, cells targeting the ATP1A1 locus and testing for small molecules or culture conditions were treated with 1 mM ouabain in StemFit AK02N medium containing ROCK inhibitor from 48 hours after EP until day 6. The cells were then routinely maintained in StemFit AK02N medium until day 13, and the forming colonies were stained with crystal violet. The cell culture plate was placed on ice, washed with ice-cold 1X DPBS, and fixed with ice-cold methanol for 10 min, after which the cells were removed from the ice and incubated with 0.05-0.1% crystal violet (Nacalai, Cat. No. 09804-52) in 25-100% methanol for 10 min at room temperature. Excess crystal violet was removed by washing with water and the plates were photographed for manual colony counting.

[0165] (Clonal analysis) For characterization of DNA repair outcomes in single clones, cells were harvested on day 7 after EP and 5 × 10 5 The cells used were 5 × 10 resuspended in STEM-CELLBANKER GMP grade (Takara Bio, Cat. No. CB047). 5 Two cryogenic tubes with 100 cells each were frozen for total population storage, and 300–600 cells were plated onto 6 cm dishes coated with iMatrix-511. Ten days after plating, colonies with a diameter of approximately 1 mm were picked under a microscope into 5 μl of medium and transferred to a 96-well PCR plate. Then, 10 μl of QuickExtract DNA Extraction Solution (Epicentre, Cat. No. QE09050) was added, and the plate was incubated at 65°C for 6 min, then at 98°C for 2 min, and stored at -30°C.

[0166] (Genotyping) For genome extraction, the density is 0.5–1 × 10 6The cells were washed with 1X DPBS, DNA was purified using DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506) as recommended in the manufacturer's instructions, and the purified DNA was resuspended in 100 μl water. Target sequences were amplified using KAPA HiFi HS ReadyMix (Kapa Biosystems, Cat. No. KK2602) polymerase chain reaction (PCR) according to the manufacturer's instructions, enzymatic PCR product purification was performed with ExoSAP-IT Express reagent (Thermo Fisher Scientific, Cat. No. 75001), and Sanger sequencing was performed using BigDye Terminator v3.1 CS Kit (Thermo Fisher Scientific, Cat. No. 4337456). Genotyping primers are listed in Tables 5 and 6. The reactions were then purified by ethanol precipitation and acquired on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence alignments were analyzed with Snapgene (GSL Biotech LLC), and sequence trace files with low-confidence base calls were excluded from the analysis.

[0167] [Table 5]

[0168] [Table 6]

[0169] (RFLP analysis) The ssODN-mediated modification of the GFP target sequence was confirmed by restriction fragment length polymorphism (RFLP) of the newly created silent restriction enzyme site. The target sequence was amplified from purified gDNA using KAPA HiFi HS ReadyMix, and the amplified products were then cleaved with FastDigest NcoI (Thermo Fisher Scientific, Cat. No. FD0573) restriction enzyme in FastDigest Buffer (Thermo Fisher Scientific, Cat. No. B64) and incubated at 37°C for 30 min. The cleaved amplified products were then separated by gel electrophoresis, acquired on an AE-9000N E-Graph (Atto) gel documentation system, and quantified with ImageJ software.

[0170] (TIDE analysis) TIDE analysis was performed on the mixed sequences using the online tool at https: / / tide.nki.nl / 71. Sequence data from 409B2-derived heterozygous GFP iPS cells were used as reference. The deletion size window was extended to 50 bp to visualize larger deletions, while other parameters were left at default.

[0171] (Fluorescence microscopy) Cells plated on iMatrix-511-coated 6-well plates in StemFit AK02N medium were directly imaged using an LSM 710 (ZEISS) confocal microscope and LSM software ZEN 2009 (ZEISS). Images were acquired with a ×10 objective and GFP and DAPI filters at appropriate gains and exposure times.

[0172] [result] (Fluorescent DNA repair assay in human iPS cells) Fluorescent reporters provide a way to visualize and quantify gene editing outcomes. GFP and BFP share high sequence homology, and a single amino acid change from tyrosine to histidine (Y66H) in the fluorophore region is sufficient to convert GFP to BFP fluorescence42. Following our previously published methods, we generated iPS cell lines heterozygously targeted at the AAVS1 locus with a stably expressing GFP fluorescent reporter in the 1383D6 parental genetic background (Figure 8A-C). The Cas9-mediated DSB in GFP was repaired using a 100 bp ssODN template designed to make four nucleotide changes that resulted in the conversion of GFP to BFP (Y66H), the insertion of a PAM blocking mutation to stabilize the BFP protein (T65S) by preventing Cas9 re-cleavage, and the construction of an NcoI restriction enzyme site for genotyping by restriction fragment length polymorphism (RFLP) (Figure 1A).

[0173] Three possible fluorescence outcomes reflect DNA repair selection: BFP, loss of fluorescence (△), and GFP correspond to HDR, MutEJ, and unmodified alleles, respectively (Figure 1B). Due to the uniform expression of the reporter from the AAVS1 locus, these three outcomes could be clearly distinguished and quantified after nuclease treatment (Figures 1C-E, 9A-C).

[0174] (Cell cycle control favors HDR outcomes) Considering the link between the cell cycle and DNA repair pathway selection, we aimed to synchronize cells using various culture conditions and chemical inhibitors. We have shown that mild cold leads to cell cycle arrest and slow cell metabolism in vitro44. We observed that cold shock at 32°C for 48 h immediately after electroporation (EP) improved HDR frequency by 1.4-fold (30.1% vs. 21.3%) and HDR / MutEJ ratio by 1.6-fold compared to normal culture conditions at 37°C, confirming our previously published results (Figures 2A, 2B, 10A, 10B).

[0175] To elucidate the effect of cold shock on iPS cells, we performed cell cycle analysis using 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay combined with propidium iodide (PI) staining on total DNA to quantify cell cycle after EP and cold shock treatment (Figure 2C). Cells that experienced cold shock for 48 h after EP showed 25.5% cell cycle accumulation in G2 / M phase compared to 6.0% under normal culture conditions. In addition, the average intensity of EdU incorporation was reduced by 2.4-fold in S-phase cells during cold shock, indicating a reduced rate of DNA synthesis. We tested the effect of cell cycle synchronization at late G2 / M phase by pretreating cells with nocodazole for 16 h before EP, but in agreement with previous reports, no increase in HDR efficiency was observed (Figures 10C-E). This would suggest that the slowdown of cell cycle progression in the presence of nuclease is more important than nuclease pretreatment for G2 / M phase synchronization. Nocodazole inhibits microtubule polymerization and activates the spindle assembly checkpoint, arresting cells in prometaphase and presumably causing cells to immediately complete mitosis shortly after nocodazole release.

[0176] Because HDR is most active in the S / G2 phase, we investigated the effect of cell synchronization using the CDC7 inhibitor XL413, which interrupts S-phase progression and arrests karyotypically normal cells in G1 / S phase45,46. As expected, EdU / PI staining revealed an accumulation of more than 90% G1 and early S-phase cells after 24 h of treatment with XL413 after EP (Figure 2D-F). XL413 increased HDR frequency by 1.7-fold (33.7% vs. 19.4%) and HDR / MutEJ ratio by 2.2-fold compared to untreated controls. When 24 h of XL413 treatment was combined with 48 h of cold shock, HDR frequency was further increased by 2-fold (39.4% vs. 19.4%) and HDR / MutEJ ratio by 2.7-fold (0.86 vs. 0.32), resulting in HDR events being almost as frequent as MutEJ. Interestingly, pretreatment of cells with XL413 prior to EP did not improve HDR (Figures S10F-S10H). These results indicate that chemical and environmental factors that regulate cell cycle synchronization and progression can independently and synergistically influence DNA repair pathway selection toward higher HDR outcomes.

[0177] Modulation of DNA repair pathways to bias HDR outcomes To further improve precise gene editing, we treated cells with various small molecules KU-5593347, VE-82148, NU744149, Mirin50, PFM0151, TDRL-50552, and SCR753, which are reported to inhibit certain DNA repair pathway components, or small molecules RS-154 and L75550755, which are reported to increase HDR ratios (Figure 3A). Human iPS cells were treated with small molecules 4 h before EP and 48 h after EP in addition to a 48-h cold shock. Among the compounds screened, NU7441 (a DNA-PKcs inhibitor) and SCR7 (a ligase IV inhibitor) increased HDR frequency by 1.6- and 1.4-fold (35.5% and 32.3% vs. 22.6%, Figure 3B), and these two compounds were the only ones with HDR / MutEJ ratios above 0.5. Interestingly, compared to NU7441, samples treated with SCR7 also exhibited a larger proportion of MutE, resulting in a reduced increment in the HDR / MutEJ ratio (1.4-fold vs. 2.2-fold in NU7441) despite a similar proportion of HDR ( Figure 3C ).

[0178] When combined, NU7441+SCR7(N+S) further improved HDR frequency by 1.8-fold (49.6% vs. 27.3%) and HDR / MutEJ ratio by 2.7-fold (1.11 vs. 0.41) compared to untreated controls, resulting in a majority of HDR events and a cumulative effect (Figure 3D, E). Meanwhile, previously reported HDR enhancers such as RS-1 and L755507 showed only minor changes in HDR frequency. These data suggest that inhibiting several molecular targets in the NHEJ repair pathway can have additive effects, further encouraging the use of HDR for DSB repair.

[0179] We next combined chemical cell cycle synchronization using XL413 with NHEJ inhibition using N+S treatment to confirm the possible additive effect of improving HDR ratio (Figure 3F-J). Under normal culture conditions, treatment with XL413 (XL) alone or N+S alone increased HDR frequency by 1.8- and 2.3-fold (30.5% and 39% vs. 17%), respectively, compared to untreated controls, whereas combining XL413 with N+S further increased HDR frequency by 2.7-fold (45.7% vs. 17%). Under cold shock conditions, comparable changes were observed in HDR frequency for the single treatments, but the combination of XL413 with N+S showed no improvement in HDR frequency compared to N+S treatment alone. In brief, we provide evidence for synergistic gene editing that enhances the frequency of HDR outcomes when combining modulation of DNA repair with cold shock or modulation of DNA repair with cell cycle synchronization at G1 / S phase.

[0180] (Highly efficient biallelic gene editing) In diploid iPS cells, both paternal and maternal alleles are editing candidates. To recapitulate this scenario, we generated homozygous GFP reporter iPS cell lines in the 1383D6 paternal genetic background (Figure 8A-C) and evaluated biallelic gene targeting outcomes (Figure 4A). We first compared the single- and biallelic targeting efficiency and DNA repair outcome frequency between heterozygous and homozygous GFP reporter lines (Figure 4B) and found comparable HDR frequencies in both lines (24.3% and 29.2%). In the homozygous context, we gated cells with at least one BFP allele as HDR outcome and cells with at least one GFP allele as non-modified, resulting in a reduced proportion of MutEJ cells and a 1.4-fold higher HDR / MutEJ ratio compared to heterozygous cell lines (Figure 4C). Strikingly, HDR *In the double-positive group, denoted as heterozygous mutants with one HDR allele (BFP) and one unmodified allele (GFP) (Fig. 4A), none were detectable. To further elucidate the cause of this, we deconvolved all possible biallelic repair outcomes and estimated the respective allelic repair efficiency (Fig. 4D). Here, we obtained 16.1% correct BFP / BFP biallelic editing and a significant proportion of MutEJ alleles distributed across BFP / △ (13.1%), △ / △ (44%), and GFP / △ (2.1%) repair outcomes. In summary, the data revealed that both alleles were largely co-edited or unmodified, as indicated by the low frequency of single alleles in the GFP / △ (2.1%) and BFP / GFP (0.3%) repair outcomes.

[0181] We further subjected homozygous GFP strains to NU7441, SCR7, and N+S under cold shock treatment and observed a 4-fold increase in HDR frequency (48.9% vs. 12.2%) and a 3.7-fold increase in HDR / MutEJ ratio (1.73 vs. 0.49) compared to untreated controls (Figure 4E, F). Deconvolution of biallelic repair outcomes showed a 30.5% BFP / BFP biallelic repair outcome using N+S treatment, 5.7-fold higher than untreated conditions (5.3%) (Figure 4G). These results demonstrated highly efficient biallelic targeting when cold shock treatment was combined with DNA repair pathway inhibitors, resulting in the effective production of homozygous mutants containing single-base alterations. Interestingly, under the conditions we tested, the percentage of heterozygous BFP / GFP outcomes was consistently low (0.7% and 0.5% with and without N+S treatment), indicating that even though homozygous outcomes were frequent, alternative targeting strategies would be required to efficiently generate heterozygous mutants.

[0182] (Creating heterozygous mutations) To address the low single-allelic editing rate while retaining a high overall HDR frequency, we employed a mixed ssODN repair template strategy. Here, we exploited the high biallelic modification rate to create HDR-mediated compound heterozygous mutations consisting of one edited allele with a silent blocking mutation and one protected allele, thereby preventing subsequent Cas9 re-cutting and indel formation at the target site (Figure 5A). In conjunction with the Y66H mutant ssODN (ssODN'M'), we designed an ssODN with a silent T65T blocking mutation (ssODN'B') and a wild-type (WT) ssODN matching the GFP target sequence (ssODN'W') as a control. HDR-mediated editing of T65T with ssODN B disrupts the PAM sequence at the target site, resulting in a GFP allele (pGFP) protected from Cas9 (Figure 11A, Figure 11B). The allele incorporating ssODN W is expected to be prone to re-cutting. Biallelic HDR repair outcomes were homozygous BFP / BFP and pGFP / pGFP cells, as well as HDR * This is expected to result in the production of a small number of desired compound heterozygous BFP / pGFP cells, referred to as

[0183] Single ssODN templates and ssODN combinations were tested (Figure 5B, Figure 5C). Compared to ssODN M (39.1%), ssODN B showed a 1.5-fold increase in GFP-positive cells (57.2%), suggesting the production of pGFP alleles by HDR, while ssODN W showed a 1.8-fold decrease (22.2%), suggesting that the WT GFP alleles produced through HDR were susceptible to re-cleavage as expected. The combination of ssODN M+B and ssODN M+W produced similar total repair frequencies. Nevertheless, among all ssODN conditions, only the combination of ssODN M and ssODN B resulted in a group of heterozygous BFP / GFP double positive cells with an efficiency of 3.6% (Figure 5D, Figure 5E), which was 12-fold higher than using ssODN M alone (0.3%) and 20-fold higher than using the combination of ssODN M and ssODN W (0.18%). When subjected to N+S treatment in addition to cold shock, the efficiency of composite heterozygous BFP / pGFP production retained a similar fold accumulation to ssODN M alone (12.4-fold) or ssODN M+W (16.2-fold) alone, but increased to 11.2% for the whole group (Figure 11C-Figure 11F). These results suggest that under high HDR efficiency conditions, protection against Cas9-mediated re-cutting is required in one allele to obtain a heterozygous mutant allele without mutagenic indels in the second allele.

[0184] As a result, we confirmed the applicability of this approach to endogenous loci. First, we targeted ATP1A1 (Figure S1A), which is known to cause a 2000-fold increase in primary cell resistance to the cytotoxic inhibitor ouabain when introducing Q118R and N129D missense mutations compared to in-frame insertion deletion mutations56. When selecting HDR clones under high ouabain concentrations, we observed a 1.8-fold increase in colony number in combination with cold shock and N+S treatment, indicating a synergistic increase in frequency at one endogenous locus (Figure S1B). Next, we aimed to edit non-selectable endogenous loci. We independently generated the N588K (c.1764C>A) mutation in KCNH2 and the G201V (c.602 G>T) mutation in PSMB8 using either ssODN with a mutation (ssODN M) or a combination of ssODN M and ssODN with a silent blocking mutation (ssODN M+B) and performed clonal analysis (Figures 12C, 12D). When using only KCNH2 or PSMB8 ssODN M, we obtained 14 out of 96 homozygous clones edited with KCNH2 and 18 out of 91 homozygous clones edited with PSMB8 under optimal cold shock and drug treatments corresponding to biallelic HDR events (Figures 12E, 12F). Furthermore, we were only able to obtain compound heterozygous clones in KCNH2 (4 / 92) or PSMB8 (4 / 95) when using ssODN M+B combinations, corresponding to biallelic HDR events incorporating mutant ssODN M and silently interrupted ssODN B in the cognate allele. These results confirmed that our approach was highly effective in generating both homozygous and heterozygous clones at endogenous loci in human iPS cells.

[0185] Synergistic gene editing promotes HDR at endogenous loci Finally, considering the application of gene-edited iPS cells for cell therapy, we analyzed our defined conditions using a GMP cell-approved gene transfer device. We compared DNA repair outcome frequencies in normal culture conditions, cold shock conditions, and cold shock combined with N+S conditions in heterozygous and homozygous GFP iPS cell lines generated in two different donor genetic backgrounds (Figures S13A-S13H). In the 1383D6 genetic background, HDR efficiency was increased by 1.2-fold with cold shock and 1.6-fold with cold shock combined with N+S treatment compared to untreated controls, both in heterozygous (59.1% and 75.6% vs. 47.9%) and homozygous (64.6% and 84.1% vs. 52.9%) cell lines. When homozygous GFP iPS cells were edited with ssODN M and ssODN B, the efficiency of composite heterodimer BFP / pGFP editing increased 1.5-fold with cold shock and 2.5-fold with the combination of cold shock and N+S treatment (14.4% to 24.1% vs. 9.8%). Similar results were obtained in the 409B2 genetic background.

[0186] Furthermore, synchronization with XL413 and DNA repair modulation with N+S treatment also showed evidence of synergistic gene editing enhancing HDR frequency (Figure 6A-6H). Remarkably, HDR outcomes reached 83.3% during single-allelic editing of heterozygous GFP iPS cells when combined with XL413 and N+S treatment under cold-shock conditions (Figure 6A, Figure 6B) and 96.6% during biallelic editing of homozygous GFP iPS cells (Figure 6C, Figure 6D, Figure 14A, Figure 14B), including 84.8% biallelic HDR editing outcomes. Furthermore, when homozygous GFP iPS cells were edited with a mixed repair template of ssODN M and ssODN B, 32.2% of cells became compound heterozygotes (Figure 6E, Figure 6F, Figure 14C, Figure 14D). Finally, we confirmed the HDR frequency of synergistic gene editing at endogenous loci using XL413 in combination with N+S (XL+N+S) or cold shock in combination with N+S (32°C+N+S) (Figure 6G, Figure 6H) compared to baseline HDR levels of untreated (-) (Figure 15A-J). HDR outcomes included clones with template-mediated repair events at one or both alleles, while MutEJ outcomes included clones with indels at at least one allele. Overall, synergistic gene editing resulted in a several-fold increase in HDR frequency at all targeted loci, supporting the broad applicability of this strategy to target the human genome (Figure 6G). We obtained 18–23 clones out of 32 clones with HDR alleles under XL+N+S treatment, implying total HDR efficiency of 56%–72% at five loci (KCNH2 N588D / N588K, APRT M136T, HES7 R25W, and PSMB8 G201V). Interestingly, cell cycle arrest in XL413 had a stronger effect on HDR ratios than cold shock when combined with N+S treatment. At the other five loci (KCNE1 D85N, KCNH2 N45D, SCN5A A1428S, and KCNJ11 T293N / T294M), total editing efficiency was low, as indicated by a higher percentage of unmodified wild-type clones, suggesting poor gRNA activity.In this case, 0-8 of 32 HDR clones, or 0-25% HDR efficiency, were achieved. Similarly, HDR / MutEJ ratios improved with synergistic gene editing, with stronger effects observed in XL413 compared to cold shock in most cases (Figure 6H). In summary, these results confirm that synergistic gene editing via cell cycle synchronization and DNA repair pathway modulation is transferable across electroporation devices and to other iPS cell lines, resulting in efficient clonal production of homozygous and compound heterozygous mutations at multiple endogenous loci.

[0187] (Consideration) In this study, we attempted to bias DNA repair outcomes toward HDR during ssODN-mediated gene editing in human iPS cells using chemical and culture condition interventions (Figure 7). Cold shock has been shown to have various effects on cellular functions, including metabolic slowing, activation of apoptotic pathways, altered gene expression, and cell cycle arrest, at severe (4–16°C), moderate (16–25°C), and mild (25–35°C) low temperatures. Moderate cold shock at 32°C is characterized by peak levels of cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3), which have important functions in cellular protection against various endogenous and environmental stresses. From the perspective of gene editing, cold shock has been shown to improve HDR efficiency, although the mechanism is unclear. Here, we show that cold shock delayed cell cycle progression, accumulated cells in the G2 / M phase, and reduced DNA synthesis rates. Alternatively, cold shock may be manifested through other cell cycle independent effects such as nuclease stability, gRNA-associated or DNA cleavage kinetics, repair intermediate stabilization, post-translational regulation stabilization, and cell viability stabilization. These additional effects of cold shock could explain the synergistic effect observed in combination with XL413-induced cell cycle arrest at G1 / S phase.

[0188] In the area of ​​DNA repair and gene editing, cell cycle regulation is an ongoing area of ​​interest. It has been proposed that increased CDK activity during S phase stimulates phosphorylation of key HDR proteins such as CtlP, promoting DSB end resection and HDR. Cell cycle synchronization at the G1 / S boundary with aphidicolin and late G2 / M with nocodazole has been shown to enhance HDR frequency. Furthermore, it has been reported that co-promotion of the G1 / S transition by cyclin D1 (CCND1) overexpression and cell cycle synchronization during G2 / M with nocodazole further improves HDR efficiency. Here we report the promotion of gene editing by the CDC7 inhibitor XL413 in iPS cells. Consistent with our observations in iPS cells, XL413 has been reported to accumulate cells in S / G2 / M phase in cancer cell lines46, whereas arrest in early G1 / S phase has been reported in karyotypically normal cells45. Human iPS cells, like embryonic stem (ES) cells, are also known to have a shortened G1 phase of about 2.5 hours and enter S phase about 4 hours after nocodazole-induced synchronization at G2 / M phase. During CRISPR-Cas9 targeting, RNP activity begins within 4 hours of delivery and is mostly terminated by RNP degradation about 24 hours later, leaving only a limited time window for DSB formation at S / G2 phase and recruitment of the HDR pathway. Thus, cell cycle delay at G2 / M or cell cycle synchronization at early G1 / S phase when nuclease activity is high may ensure rapid cell cycle transition to S phase, which is in any case the dominant cell cycle state in iPS cells and favors HDR repair. It is intriguing that nocodazole failed to promote iPS cell gene editing in our experiments, but XL413 had a clear positive effect that warrants further exploration.

[0189] We found that the NHEJ inhibitors NU7441 and SCR7 independently improved HDR efficiency in iPS cells and further improved HDR efficiency when combined. DNA-PKcs recruits protein components of the NHEJ pathway, including DNA ligase IV, which is specifically activated upon DSB formation and acts to join DNA ends. We speculate that inhibition of DNA-PKcs with NU7441 may avoid NHEJ complex formation and select alternative DNA repair pathways, whereas downstream inhibition of DNA ligase IV with SCR7 may already commit cells to NHEJ repair or some alternative MutEJ repair. This could explain the higher HDR efficiency with NU7441 compared to SCR7 treatment. Interestingly, no promotion of HDR by RS-1 or L755507 was observed. Regarding the particular type of HDR that we aimed to induce, recent reports speculate that ssODN-mediated DSB repair undergoes synthesis dependent strand annealing (SDSA), a RAD51-independent process, thus explaining the limited effect of the HDR enhancers, RS-1 and possibly L755507, in our system.

[0190] Previous studies that attempted to combine cell cycle synchronization with NHEJ inhibitors, however, did not observe an improvement in HDR efficiency compared to either condition alone. This could be due to the use of karyotypically abnormal cell lines such as HEK293T cells or the low efficiency of SCR7 inhibitors alone in pluripotent stem cells. In addition, HDR activity peaks in mid-S phase and declines in G2 phase in karyotypically normal cells, which may explain the limited efficiency we observed in iPS cells upon late G2 / M phase synchronization with nocodazole. Taken together, these results indicate that alternative conditions that simultaneously regulate cell cycle and DNA repair pathway selection synergistically improve HDR frequency. To our knowledge, we are the first to report the synergistic effect of cell cycle synchronization and DNA repair pathway modulation on precise gene editing.

[0191] Using this approach, we obtained cells with up to 96.6% HDR alleles during biallelic editing, including 84.8% biallelic HDR editing when homozygous mutations were created. Strikingly, high editing efficiency was associated with almost absent heterozygous mutant cells. Consistent with previous reports, single HDR alleles formed by precise single-allelic editing were mostly paired with indels in the second allele.

[0192] Therefore, we employed a mixed ssODN strategy to form compound heterozygous mutant alleles protected from re-cutting by Cas9, producing up to 32.2% compound heterozygous mutants in a GFP reporter. Given the endogenous targets for disease models, we created homozygous and compound heterozygous mutations at multiple loci and demonstrated that synergistic gene editing consistently improved HDR outcome frequency several-fold compared to baseline HDR levels. Ultimately, obtaining heterozygous mutations without silent mutations protecting the second allele may require a second round of editing, or the use of two-step donor vector targeting and excision processes such as the MhAX method. Other strategies could leverage haplotype specificity, conversion systems, or nuclease titration to bias toward single-allele editing, but these approaches would be locus-dependent and would come at the cost of low editing efficiency. We expect that synergistic gene editing will improve ssODN-based strategies that utilize two-step targeting to introduce and remove blocking mutations. In conclusion, we established a biallelic reporter system capable of resolving allele-specific DNA repair outcomes and defined synergistic gene editing conditions that utilize cold shock, cell cycle synchronization, and DNA repair modulation to improve HDR and HDR / MutEJ ratios. Taking advantage of highly efficient biallelic modifications, we demonstrated a strategy to generate compound heterozygous iPS cell lines. We anticipate that improving the reliability of biallelic editing outcomes will greatly facilitate the creation of overt and recessive genetic disease models and potentially even the establishment of treatments using human iPS cells. [Industrial Applicability]

[0193] According to the present invention, a technique can be provided for efficiently producing genome-edited cells in which targeted DNA double-strand breaks (DSBs) in the genome are repaired by the homology-directed repair (HDR) pathway or an equivalent pathway.

Claims

1. A method for producing genome-edited cells (excluding cells present in a human body, human germ cells, and human embryonic cells), comprising the steps of: synchronizing the cell cycle of said cells; treating the cells with an agent that alters a DNA repair pathway; introducing donor DNA into said cells; Inducing a sequence-specific double-strand break in the genomic DNA of the cell in the presence of the donor DNA, so that the double-strand break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, thereby obtaining the cell whose genome has been edited; Including, synchronizing the cell cycle of the cells comprises incubating the cells at 25° C. to 35° C. for 24 to 72 hours and treating the cells with XL413 (CAS No: 1169558-38-6); A method for producing a genome-edited cell, wherein the agent that alters a DNA repair pathway includes NU7441 (CAS number: 503468-95-9) and SCR7 (CAS number: 14892-97-8).

2. The sequence-specific double-stranded break is induced by a ribonucleoprotein (RNP) complex comprising Cas9 and gRNA; The method of claim 1 .

3. The donor DNA is a single-stranded donor oligonucleotide. The method according to claim 1 or 2.

4. The donor DNA has a nucleotide sequence that modifies a protospacer sequence or a protospacer adjacent motif (PAM) sequence; The method according to claim 3.

5. The donor DNA has a mutation, and a cell is obtained in which the mutation has been introduced into one or both alleles. The method according to any one of claims 1 to 4.

6. The donor DNA includes a combination of multiple types of donor DNA corresponding to the same region on the genome. The method according to any one of claims 1 to 5.

7. the donor DNA comprises a combination of a first donor DNA and a second donor DNA; the first donor DNA has a mutation of interest and the second donor DNA has a mutation that alters a protospacer sequence or a PAM sequence; The mutation of interest is introduced into only one allele, resulting in a cell in which the protospacer sequence or PAM sequence of the other allele is modified. The method according to claim 6.

8. The cells are induced pluripotent stem (iPS) cells; The method according to any one of claims 1 to 7.

9. Cells (excluding cells present in the human body, human germ cells, and human embryonic cells) A method for editing the genome of a synchronizing the cell cycle of said cells; treating the cells with an agent that alters a DNA repair pathway; introducing donor DNA into said cells; Inducing a sequence-specific double-strand break in the genomic DNA of the cell in the presence of the donor DNA, such that the double-strand break is repaired by homologous recombination or an equivalent pathway based on all or a portion of the base sequence of the donor DNA, thereby obtaining the cell having an edited genome; Including, synchronizing the cell cycle of the cells comprises incubating the cells at 25° C. to 35° C. for 24 to 72 hours and treating the cells with XL413; A method of editing the genome of a cell, wherein the agents that alter DNA repair pathways include NU7441 and SCR7.

10. A method for detecting genome editing on one or both alleles, comprising: introducing donor DNA into a cell (excluding cells present in a human living body, human germ cells, and human embryonic cells), the cell having a gene encoding a fluorescent protein that emits a first fluorescence in both alleles, the donor DNA having a mutation that changes the first fluorescence of the fluorescent protein to a second fluorescence; Inducing a sequence-specific double-strand break in the genomic DNA of the cell in the presence of the donor DNA, so that the double-strand break is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, thereby obtaining a genome-edited cell; Analyzing the fluorescence of the genome edited cells; and As a result, (i) the first fluorescence is detected and the second fluorescence is not detected; (i-1) both alleles of the fluorescent protein locus were unchanged; or (i-2) one allele of the gene locus of the fluorescent protein is unchanged, and an insertion or deletion mutation has occurred in the other allele; (ii) if the first fluorescence is not detected and the second fluorescence is detected, (ii-1) both alleles of the fluorescent protein locus have been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or part of the base sequence of the donor DNA; or (ii-2) one allele of the fluorescent protein locus is repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, and an insertion mutation or deletion mutation has occurred in the other allele; (iii) if neither the first nor the second fluorescence is detected, this indicates that an insertion or deletion mutation has occurred in both alleles of the fluorescent protein locus; (iv) when both the first fluorescence and the second fluorescence are detected, it indicates that one allele of the fluorescent protein locus has been repaired by a homologous recombination repair pathway or an equivalent pathway based on all or a part of the base sequence of the donor DNA, and the other allele has not changed; A method for detecting genome editing on one or both alleles.

11. the fluorescent protein emitting a first fluorescence is a green fluorescent protein (GFP), the fluorescent protein emitting a second fluorescence is a blue fluorescent protein (BFP), and the donor DNA has a mutation that substitutes a tyrosine residue at position 66 of GFP with a histidine residue to modify the GFP into BFP; The method of claim 10.

12. A genome editing kit comprising XL413, NU7441, and SCR7.

13. Further comprising a sequence-specific DNA cleavage enzyme and donor DNA; A genome editing kit as described in claim 12.

14. The donor DNA has a mutation that replaces the 66th tyrosine residue of GFP with a histidine residue to modify it into BFP. A genome editing kit as described in claim 13.

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