Composition for cleaving target DNA comprising guide RNA specific for target DNA and cas protein-encoding nucleic acid or cas protein, and use thereof

Abstract

To provide a method for producing mammals having engineered mammalian cells in targeted genome editing in eukaryotic cells or organisms.SOLUTION: A method for producing mammals having engineered mammalian cells, including administering one or more engineered mammalian cells to the mammal, where the engineered mammalian cells comprise: a nucleus containing an at least partially disrupted target DNA; one or more Cas9 / RNA complexes, where each Cas9 / RNA complex comprises: a Cas9 protein and a guide RNA having a CRISPR RNA (crRNA) and a transcription-activating crRNA (tracrRNA), where the crRNA comprises i) a first portion that hybridizes to a portion of a racrRNA and ii) a second portion that is complementary to the target DNA, where at least one of the Cas9 / RNA complexes is present in the nucleus, and where the engineered mammalian cell does not comprise plasmid DNA.SELECTED DRAWING: Figure 1a

Description

[Technical field]

[0001] The present invention relates to targeted genome editing in eukaryotic cells or organisms.More particularly, the present invention relates to a composition for cleaving target DNA in eukaryotic cells or organisms, comprising target DNA-specific guide RNA and Cas protein-encoding nucleic acid or Cas protein, and its use. [Background technology]

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic loci that contain multiple short tandem repeats found in approximately 40% of sequenced bacterial and 90% of sequenced archaeal genomes. CRISPR functions as an immune system in prokaryotes, where it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a type of acquired immunity. Short segments of foreign DNA, called spacers, are integrated between the CRISPR repeats in the genome and act as a memory of past exposure. The CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner similar to RNAi in eukaryotes.

[0003] Cas9, an essential protein component of the type II CRISPR / Cas system, forms an active endonuclease when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby cleaving foreign genetic elements in invading phages or plasmids to defend the host cell. The crRNA is transcribed from a CRISPR element in the host genome that was previously captured from such a foreign invader. Recently, Jinek et al. (1) demonstrated that a single-stranded chimeric RNA produced by fusion of the essential parts of the crRNA and tracrRNA can replace the two RNAs in the Cas9 / RNA complex and form a functional endonuclease.

[0004] The CRISPR / Cas system offers an advantage over zinc finger and transcription activator-like effector DNA-binding proteins because site specificity in nucleotide-binding CRISPR-Cas proteins is governed by an RNA molecule instead of a DNA-binding protein, which can be more difficult to design and synthesize. However, until now, no genome editing method using RNA-guided endonucleases (RGENs) based on the CRISPR / Cas system has been developed. On the other hand, restriction fragment length polymorphism (RFLP) is one of the oldest, most convenient and least expensive genotyping methods that is still widely used in molecular biology and genetics, but is often limited by the lack of suitable sites recognized by restriction endonucleases.

[0005] Engineered nuclease-induced mutations are detected by a variety of methods, including the mismatch-sensitive T7 endonuclease I (T7E1) assay or Surveyor nuclease assay, RFLP, capillary electrophoresis of fluorescent PCR products, dideoxy sequencing, and deep sequencing. The T7E1 and Surveyor assays are widely used but cumbersome. Furthermore, these enzymes tend to underestimate mutation frequencies because mutant sequences may form homoduplexes with each other and cannot distinguish homozygous biallelic mutant clones from wild-type cells. RFLP is the preferred method because it does not have these constraints. In fact, RFLP was one of the first methods for detecting engineered nuclease-mediated mutations in cells and animals. Unfortunately, RFLP is limited by the availability of suitable restriction enzyme recognition sites. There may be no available restriction enzyme recognition sites at the desired target site. Summary of the Invention [Problem to be solved by the invention]

[0006] To date, no genome editing and genotyping methods using RNA-guided endonucleases (RGENs) based on the CRISPR / Cas system have been developed. Under these circumstances, the present inventors have made great efforts to develop a genome editing method based on the CRISPR / Cas system, and finally established a programmable RNA-guided endonuclease that can target and cleave DNA in eukaryotic cells and organisms. In addition, the present inventors have made great efforts to develop a novel method for using RNA-guided endonuclease (RGEN) in RFLP analysis. The present inventors have used RGEN to genotype recurrent mutations found in cancer and mutations induced in cells and organisms by artificial nucleases, including RGEN itself, thereby completing the present invention. [Means for solving the problem]

[0007] An object of the present invention is to provide a composition for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA specific to the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. Another object of the present invention is to provide a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific to a target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0008] Yet another object of the present invention is to provide a kit for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. Yet another object of the present invention is to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0009] It is yet another object of the present invention to provide a method for producing a eukaryotic cell or organism having a Cas protein and a guide RNA, comprising co-transfecting or sequentially transfecting a Cas protein-encoding nucleic acid or a Cas protein, and a guide RNA or a DNA encoding the guide RNA into the eukaryotic cell or organism. Yet another object of the present invention is to provide a eukaryotic cell or organism containing a guide RNA or a DNA encoding a guide RNA specific to a target DNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0010] It is yet another object of the present invention to provide a method for cleaving a target DNA in a eukaryotic cell or organism, comprising the step of transfecting a composition containing a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein into the eukaryotic cell or organism containing the target DNA. It is yet another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism, comprising treating the eukaryotic cell or organism with a composition containing a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein.

[0011] It is yet another object of the present invention to provide an embryo, a genome-modified animal, or a genome-modified plant having a genome edited by a composition containing a guide RNA specific for a target DNA or a DNA encoding a guide RNA and a Cas protein-encoding nucleic acid or a Cas protein. It is yet another object of the present invention to provide a method for producing a genome-modified animal, comprising the steps of: introducing a composition containing a guide RNA specific to a target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein into an animal embryo; and transferring the embryo into the oviduct of a pseudopregnant surrogate mother to give rise to a genome-modified animal.

[0012] Yet another object of the present invention is to provide a composition for genotyping mutations or variations in an isolated biological sample, comprising a guide RNA specific for a target DNA sequence and a Cas protein. Yet another object of the present invention is to provide a method for using RNA-guided endonucleases (RGENs) to genotype mutations induced in cells by artificial nucleases or naturally occurring mutations or polymorphisms, the RGENs comprising a guide RNA specific for a target DNA and a Cas protein.

[0013] Yet another object of the present invention is to provide a kit for genotyping an artificially induced mutation or a naturally occurring mutation or polymorphism in a cell, comprising an RNA-guided endonuclease (RGEN), the RGEN comprising a guide RNA specific for a target DNA and a Cas protein. An object of the present invention is to provide a composition for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA specific to the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0014] Another object of the present invention is to provide a composition for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific to a target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. Yet another object of the present invention is to provide a kit for cleaving a target DNA in a eukaryotic cell or organism, comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0015] Yet another object of the present invention is to provide a kit for inducing targeted mutagenesis in eukaryotic cells or organisms, comprising a guide RNA specific for a target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. It is yet another object of the present invention to provide a method for producing a eukaryotic cell or organism having a Cas protein and a guide RNA, comprising co-transfecting or sequentially transfecting a Cas protein-encoding nucleic acid or a Cas protein, and a guide RNA or a DNA encoding the guide RNA into the eukaryotic cell or organism.

[0016] Yet another object of the present invention is to provide a eukaryotic cell or organism containing a guide RNA or a DNA encoding a guide RNA specific to a target DNA, and a Cas protein-encoding nucleic acid or a Cas protein. It is yet another object of the present invention to provide a method for cleaving a target DNA in a eukaryotic cell or organism, comprising the step of transfecting a composition containing a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein into the eukaryotic cell or organism containing the target DNA.

[0017] It is yet another object of the present invention to provide a method for inducing targeted mutagenesis in a eukaryotic cell or organism, comprising treating the eukaryotic cell or organism with a composition containing a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein. It is yet another object of the present invention to provide an embryo, a genome-modified animal, or a genome-modified plant having a genome edited by a composition containing a guide RNA specific for a target DNA or a DNA encoding a guide RNA and a Cas protein-encoding nucleic acid or a Cas protein.

[0018] It is yet another object of the present invention to provide a method for producing a genome-modified animal, comprising the steps of: introducing a composition containing a guide RNA specific to a target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein into an animal embryo; and transferring the embryo into the oviduct of a pseudopregnant surrogate mother to give rise to a genome-modified animal. Yet another object of the present invention is to provide a composition for genotyping mutations or polymorphisms in an isolated biological sample, comprising a guide RNA specific for a target DNA sequence and a Cas protein.

[0019] Yet another object of the present invention is to provide a composition for genotyping a nucleic acid sequence of a pathogenic microorganism in an isolated biological sample, comprising a guide RNA specific to a target DNA sequence and a Cas protein. Yet another object of the present invention is to provide a kit for genotyping mutations or polymorphisms in an isolated biological sample, comprising a composition, specifically a composition containing an RNA-guided endonuclease (RGEN), wherein the RGEN comprises a guide RNA specific for a target DNA and a Cas protein.

[0020] Yet another object of the present invention is to provide a method for genotyping mutations or polymorphisms in an isolated biological sample using a composition, specifically a composition containing an RNA-guided endonuclease (RGEN), which comprises a guide RNA specific for a target DNA and a Cas protein. Effect of the Invention

[0021] The present composition, which contains a guide RNA specific for a target DNA and a Cas protein-encoding nucleic acid or a Cas protein for cutting a target DNA or inducing targeted mutagenesis in eukaryotic cells or organisms, a kit containing the present composition, and a method for inducing targeted mutagenesis provide a new and convenient genome editing tool.In addition, since custom RGEN can be designed to target any DNA sequence, almost any single nucleotide polymorphism or small insertion / deletion (indel) can be analyzed by RGEN-mediated RFLP, and therefore the composition and method of the present invention can be used to detect and cut naturally occurring polymorphisms and mutations. [Brief description of the drawings]

[0022] [Figure 1a]Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (a) Schematic of the target DNA and chimeric RNA sequences. Red triangles indicate the cleavage site. The PAM sequence recognized by Cas9 is shown in bold. Sequences in the guide RNAs derived from the crRNA and tracrRNA are boxed and underlined, respectively. [Figure 1b] Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (b) In vitro cleavage of plasmid DNA by Cas9. Intact circular or ApaLI-digested plasmids were incubated with Cas9 and guide RNA. [Figure 2a] Figure 2 shows Cas9-induced mutagenesis at an episomal target site. (a) Schematic of a cell-based assay using an RFP-GFP reporter. The GFP sequence is frameshifted and fused to the RFP sequence, so GFP is not expressed from this reporter. The RFP-GFP fusion protein is expressed only if the target site between the two sequences is cleaved by a site-specific nuclease. [Figure 2b] Figure 2 shows Cas9-induced mutagenesis at an episomal target site. (b) Flow cytometry of Cas9-transfected cells. The percentage of cells expressing RFP-GFP fusion protein is shown. [Figure 3a] Figure 3 shows RGEN-induced mutations at endogenous chromosomal sites. (a) CCR5 locus. (Top) RGEN-induced mutations were detected using the T7E1 assay. The arrows indicate the predicted positions of DNA bands cleaved by T7E1. Mutation frequencies (Indels (%)) were calculated by measuring band intensity. (Bottom) DNA sequences of wild-type (WT) and mutant clones of CCR5 and C4BPB. Regions of the target sequence complementary to the guide RNA are boxed (boc). PAM sequences are shown in bold. Triangles indicate the cleavage site. Bases corresponding to microhomologies are underlined. The right column indicates the number of bases inserted or deleted. [Figure 3b]Figure 3 shows RGEN-induced mutations at endogenous chromosomal sites. (b) C4BPB locus. (Top) RGEN-induced mutations were detected using the T7E1 assay. Arrows indicate the predicted positions of DNA bands cleaved by T7E1. Mutation frequencies (Indels (%)) were calculated by measuring band intensity. (Bottom) DNA sequences of wild-type (WT) and mutant clones of CCR5 and C4BPB. Regions of the target sequence complementary to the guide RNA are boxed (boc). PAM sequences are shown in bold. Triangles indicate the cleavage site. Bases corresponding to microhomologies are underlined. The right column indicates the number of bases inserted or deleted. [Figure 4a] Figure 4 shows that no RGEN-induced off-target mutations were detected. (a) On-target sequence and potential off-target sequence. A computer search was performed on the human genome for potential off-target sites. Four sites were identified, each with a three-base mismatch with the CCR5 on-target site. The mismatched bases are underlined. [Figure 4b] Figure 4 shows that no RGEN-induced off-target mutations were detected. (b) Using the T7E1 assay, we examined whether these sites were mutated in cells transfected with Cas9 / RNA complexes. No mutations were detected at these sites. N / A (not applicable), intergenic sites. [Figure 4c] Figure 4 shows that RGEN-induced off-target mutations were not detected. (c) Cas9 did not cause off-target related chromosomal deletions. CCR5-specific RGEN and ZFN were expressed in human cells. PCR was used to detect the induction of a 15-kb chromosomal deletion in these cells. [Figure 5a] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (a) Schematic diagram showing sgRNA specific for exon 2 of mouse Foxn1 gene. The PAM of exon 2 is shown in red, and the sequence in the sgRNA complementary to exon 2 is underlined. The triangle indicates the cleavage site. [Figure 5b] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (b) Representative T7E1 assay showing gene targeting efficiency of Cas9 mRNA and Foxn1-specific sgRNA delivered to one-cell stage mouse embryos by intracytoplasmic injection. Numbers indicate independent founder mice resulting from the highest dose. Arrows indicate bands cleaved by T7E1. [Figure 5c] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (c) DNA sequences of mutant alleles observed in the three Foxn1 mutant founders identified in b. The occurrence frequency is indicated in brackets. [Figure 5d] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (d) PCR genotyping of F1 offspring resulting from mating Foxn1 founder #108 with wild-type FVB / NTac. Segregation of mutant alleles found in Foxn1 founder #108 in offspring is observed. [Figure 6] Figure 6 shows Foxn1 gene targeting in mouse embryos by intracytoplasmic injection of Cas9 mRNA and Foxn1-sgRNA. (a) Representative results of T7E1 assay measuring mutation rate after injection of the highest dose. Arrows indicate bands cleaved by T7E1. (b) Summary of T7E1 assay results. Mutation rates among in vitro cultured embryos obtained after intracytoplasmic injection of the indicated RGEN doses. (c) DNA sequences of Foxn1 mutant alleles identified from a subset of T7E1 positive mutant embryos. Target sequences of wild type alleles are boxed. [Figure 7a] Figure 7 shows Foxn1 gene targeting in mouse embryos using recombinant Cas9 protein:Foxn1-sgRNA complex. (a) and (b) are representative T7E1 assay results and their summary. After receiving pronuclear injection (a) or intracytoplasmic injection (b), the embryos were cultured in vitro. Red numbers indicate T7E1-positive mutant founder mice. [Figure 7b]Figure 7 shows Foxn1 gene targeting in mouse embryos using recombinant Cas9 protein:Foxn1-sgRNA complex. (a) and (b) are representative T7E1 assay results and their summary. After receiving pronuclear injection (a) or intracytoplasmic injection (b), the embryos were cultured in vitro. Red numbers indicate T7E1-positive mutant founder mice. [Figure 7c] Figure 7 shows Foxn1 gene targeting in mouse embryos using recombinant Cas9 protein: Foxn1-sgRNA complex. (c) DNA sequence of Foxn1 mutant allele identified from in vitro cultured embryos obtained by pronuclear injection with the highest dose of recombinant Cas9 protein: Foxn1-sgRNA complex. Target sequence of wild type allele is boxed. [Figure 8] Figure 8 shows germline transmission of the mutant allele found in Foxn1 mutant founder #12. (a) fPCR analysis. (b) PCR genotyping of wild-type FVB / NTac, founder mice, and their F1 progeny. [Figure 9] Figure 9 shows the genotypes of embryos generated by mating Prkdc mutant founders. Prkdc mutant founders ♂25 and ♀15 ​​were mated, and E13.5 embryos were excised. (a) fPCR analysis of wild type, founder ♂25, and founder ♀15. It should be noted that due to technical limitations of fPCR analysis, these results showed slight differences in the exact sequence of the mutant alleles; for example, sequence analysis identified Δ269 / Δ61 / WT and Δ5+1 / +7 / +12 / WT in founders ♂25 and ♀15, respectively. (b) Genotypes of the resulting embryos. [Figure 10a] FIG. 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10b] FIG. 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10c] FIG. 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10d] FIG. 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10e] FIG. 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 11] FIG. 11 shows recombinant Cas9 protein-induced mutations in Arabidopsis protoplasts. [Figure 12] FIG. 12 shows recombinant Cas9 protein-induced mutation sequences in the Arabidopsis BRI1 gene. [Figure 13] FIG. 13 shows a T7E1 assay demonstrating the disruption of endogenous CCR5 gene in 293 cells by treatment with Cas9-mal-9R4L and sgRNA / C9R4LC complex. [Figure 14a] Figure 14 (a, b) shows the mutation frequency at the on-target and off-target sites of RGEN reported in Fu et al. (2013). (R) T7E1 assay analyzing genomic DNA from K562 cells transfected sequentially with 20 μg of Cas9-encoding plasmid and 60 μg and 120 μg of in vitro transcribed GX19 crRNA and tracrRNA, respectively (1 × 106 cells), or (D) co-transfected with 1 μg of Cas9-encoding plasmid and 1 μg of GX19 sgRNA expression plasmid (2 × 105 cells). [Figure 14b] Figure 14 (a, b) shows the mutation frequency at the on-target and off-target sites of RGEN reported in Fu et al. (2013). (R) T7E1 assay analyzing genomic DNA from K562 cells transfected sequentially with 20 μg of Cas9-encoding plasmid and 60 μg and 120 μg of in vitro transcribed GX19 crRNA and tracrRNA, respectively (1 × 106 cells), or (D) co-transfected with 1 μg of Cas9-encoding plasmid and 1 μg of GX19 sgRNA expression plasmid (2 × 105 cells). [Figure 15a] Figure 15(a, b) shows a comparison of guide RNA structures. The mutation frequency of RGEN reported in Fu et al. (2013) was measured at on-target and off-target sites using the T7E1 assay. A plasmid encoding Cas9 and a plasmid encoding GX19 sgRNA or GGX20 sgRNA were co-transfected into K562 cells. Off-target sites (such as OT1-3) are labeled as described in Fu et al. (2013). [Figure 15b] Figure 15(a, b) shows a comparison of guide RNA structures. The mutation frequency of RGEN reported in Fu et al. (2013) was measured at on-target and off-target sites using the T7E1 assay. A plasmid encoding Cas9 and a plasmid encoding GX19 sgRNA or GGX20 sgRNA were co-transfected into K562 cells. Off-target sites (such as OT1-3) are labeled as described in Fu et al. (2013). [Figure 16a]

[0023] Figure 16 shows in vitro DNA cleavage by Cas9 nickases. (a) Schematic of Cas9 nuclease and paired Cas9 nickases. The PAM sequence and cleavage site are boxed. [Figure 16b] Figure 16 shows in vitro DNA cleavage by Cas9 nickase. (b) Target sites within the human AAVS1 locus. The location of each target site is indicated by a triangle. [Figure 16c] Figure 16 shows in vitro DNA cleavage by Cas9 nickase. (c) Schematic of the DNA cleavage reaction. FAM dye (boxed) was attached to both 5' ends of the DNA substrate. [Figure 16d] Figure 16 shows in vitro DNA cleavage by Cas9 nickase. (d) DSBs and SSBs analyzed using fluorescent capillary electrophoresis. Fluorescently labeled DNA substrates were incubated with Cas9 nuclease or nickase prior to electrophoresis. [Figure 17a]Figure 17 shows a comparison of the behavior of Cas9 nuclease and nickases. (a) On-target mutation frequencies associated with Cas9 nuclease (WT), nickase (D10A), and paired nickases. Paired nickases that would generate 5' or 3' overhangs are shown. [Figure 17b] Figure 17 shows a comparison of the behavior of Cas9 nuclease and nickase. (b) Analysis of off-target effects of Cas9 nuclease and paired nickases. A total of seven potential off-target sites for the three sgRNAs were analyzed. [Figure 18a] Figure 18 shows paired Cas9 nickases tested at other endogenous human loci. (a) sgRNA target sites at the human CCR5 and BRCA2 loci. PAM sequences are shown in red. [Figure 18b] Figure 18 shows paired Cas9 nickases tested at other endogenous human loci. (b) Genome editing activity at each target site was detected by T7E1 assay. Repair of two nicks that would have created 5' overhangs caused indel formation much more frequently than those that created 3' overhangs. [Figure 18c] Figure 18 shows paired Cas9 nickases tested at other endogenous human loci. (c) sgRNA target sites at the human CCR5 and BRCA2 loci. PAM sequences are shown in red. [Figure 18d] Figure 18 shows paired Cas9 nickases tested at other endogenous human loci. (d) Genome editing activity at each target site was detected by T7E1 assay. Repair of two nicks that would have created 5' overhangs caused indel formation much more frequently than those that created 3' overhangs. [Figure 19]FIG. 19 shows that paired Cas9 nickases mediate homologous recombination. (a) Method to detect homologous recombination. The donor DNA contained an XbaI restriction enzyme site between the two homology arms, and the endogenous target site lacked this site. A PCR assay was used to detect the homologously recombined sequences. To prevent amplification of contaminating donor DNA, primers specific for genomic DNA were used. (b) Efficiency of homologous recombination. Only the amplicon of the region where homologous recombination occurred was digested by XbaI; the intensity of the cleavage band was used to measure the efficiency of the method. [Figure 20a] Figure 20 shows DNA splicing induced by paired Cas9 nickases. (a) Target sites of paired nickases in the human AAVS1 locus. The distance between the AS2 site and each of the other sites is indicated. Arrows indicate PCR primers. [Figure 20b] Figure 20 shows DNA splicing induced by paired Cas9 nickases. (b) Genomic deletions detected using PCR. Asterisks indicate deletion-specific PCR products. [Figure 20c] Figure 20 shows DNA splicing induced by paired Cas9 nickases. (c) DNA sequence of deletion-specific PCR product obtained using AS2 and L1 sgRNA. Target site PAM sequence is boxed and sgRNA-matched sequence is in uppercase. Complete sgRNA-matched sequence is underlined. [Figure 20d] Figure 20 shows DNA splicing induced by paired Cas9 nickases. (d) Schematic model of paired Cas9 nickase-mediated chromosomal deletion. The newly synthesized DNA strand is boxed. [Figure 21a] Figure 21 shows that paired Cas9 nickases do not cause translocations. (a) Schematic of a chromosomal translocation between an on-target site and an off-target site. [Figure 21b] Figure 21 shows that paired Cas9 nickases do not cause translocations. (b) PCR amplification to detect chromosomal translocations. [Figure 21c]Figure 21 shows that paired Cas9 nickases do not cause translocation. (c) Translocation is caused by Cas9 nuclease but not by the nickase pair. [Figure 22a] Figure 22 shows a schematic diagram of the T7E1 assay and RFLP assay. (a) Comparison of assay cleavage reactions in four possible situations: (A) wild type, (B) monoallelic mutation, (C) different biallelic mutations (heterozygous), and (D) identical biallelic mutations (homozygous) after artificial nuclease treatment in diploid cells. Black lines represent PCR products derived from each allele; dashed and dotted boxes indicate insertion / deletion mutations generated by NHEJ. [Figure 22b] Figure 22 shows a schematic diagram of the T7E1 assay and RFLP assay. (b) Expected results of T7E1 digestion and RGEN digestion separated by electrophoresis. [Diagram 23] Figure 23 shows an in vitro cleavage assay of linearized plasmids containing C4BPB target sites with indels. DNA sequences of individual plasmid substrates (top). PAM sequences are underlined. Inserted bases are boxed. Arrows (bottom) indicate the expected positions of DNA bands after electrophoresis cleaved by wild-type specific RGEN. [Figure 24] Figure 24 shows the genotyping of mutations induced in cells by artificial nucleases using RGEN-mediated RFLP. (a) Genotypes of C4BPB mutant K562 cell clones. (b) Comparison of mismatch-sensitive T7E1 assay with RGEN-mediated RFLP analysis. Black arrows indicate cleavage products from T7E1 enzyme or RGEN treatment. [Figure 25a] Figure 25 shows genotyping of RGEN-induced mutations using the RGEN-RFLP method. (a) Analysis of C4BPB disrupted clones using RGEN-RFLP and T7E1 assays. Arrows indicate the expected positions of DNA bands cleaved by RGEN or T7E1. [Figure 25b]Figure 25 shows genotyping of RGEN-induced mutations using the RGEN-RFLP method. (b) Quantitative comparison of RGEN-RFLP analysis with the T7E1 assay. Genomic DNA samples from wild-type and C4BPB-disrupted K562 cells were mixed in various ratios and subjected to PCR amplification. [Figure 25c] Figure 25 shows genotyping of RGEN-induced mutations using the RGEN-RFLP method. (c) Genotyping of RGEN-induced mutations in the HLA-B gene of HeLa cells using RFLP and T7E1 analysis. [Figure 26a] Figure 26 shows the genotyping of mutations induced in organisms by artificial nucleases using RGEN-mediated RFLP. (a) Genotype of Pibf1 mutant founder mice. [Figure 26b] Figure 26 shows the genotyping of mutations induced in organisms by artificial nucleases using RGEN-mediated RFLP. (b) Comparison of mismatch-sensitive T7E1 assay with RGEN-mediated RFLP analysis. Black arrows indicate cleavage products from T7E1 enzyme or RGEN treatment. [Figure 27] Figure 27 shows RGEN-mediated genotyping of ZFN-induced mutations. The ZFN target site is boxed. The black arrow indicates the DNA band cleaved by T7E1. [Figure 28] Figure 28 shows polymorphic sites in a region of the human HLA-B gene. The sequence surrounding the RGEN target site is from a PCR amplicon from HeLa cells. The location of the polymorphism is shown in a box. The RGEN target site and the PAM sequence are shown in a dashed box and a bold box, respectively. The primer sequence is underlined. [Figure 29]Figure 29 shows genotyping of oncogenic mutations using RGEN-RFLP analysis. (a) Recurrent mutations in the human CTNNB1 gene (c.133-135 deletion of TCT) in HCT116 cells were detected by RGEN. HeLa cells were used as a negative control. (b) Genotyping of KRAS substitution mutations (c.34 G>A) in A549 cancer cell line using RGEN with mismatched guide RNA. Mismatched nucleotides are boxed. HeLa cells were used as a negative control. Arrows indicate DNA bands cleaved by RGEN. DNA sequences confirmed by Sanger sequencing are shown. [Figure 30a] Figure 30 shows the genotyping of the CCR5 delta32 allele in HEK293T cells using RGEN-RFLP analysis. (a) RGEN-RFLP assay of cell lines. K562, SKBR3, and HeLa cells were used as wild-type controls. The arrows indicate the DNA bands cleaved by RGEN. [Figure 30b] Figure 30 shows the genotyping of the CCR5 delta32 allele in HEK293T cells using RGEN-RFLP analysis. (b) DNA sequences of wild-type and delta32 CCR5 alleles. Both the on-target and off-target sites of the RGEN used in RFLP analysis are underlined. The single nucleotide mismatch between the two sites is boxed. The PAM sequence is underlined. [Figure 30c] Figure 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells using RGEN-RFLP analysis. (c) In vitro cleavage of plasmids containing WT or del32 CCR5 alleles using wild-type specific RGEN. [Figure 30d]Figure 30 shows genotyping of the CCR5 delta32 allele in HEK293T cells using RGEN-RFLP analysis. (d) Confirmation of the presence of off-target sites of CCR5-delta32-specific RGEN at the CCR5 locus. In vitro cleavage assay of plasmids containing on-target or off-target sequences using various amounts of del32-specific RGEN. [Figure 31a] Figure 31 shows the genotyping of KRAS point mutation (c.34 G>A). (a) RGEN-RFLP analysis of KRAS mutation (c.34 G>A) in cancer cell lines. PCR products from HeLa cells (used as wild-type control) or A549 cells (homozygous for the point mutation) were digested by RGEN with perfectly matched crRNA specific for the wild-type or mutant sequence. The KRAS genotype in these cells was confirmed by Sanger sequencing. [Figure 31b] Figure 31 shows genotyping of KRAS point mutation (c.34 G>A). (b) Plasmids containing either wild-type or mutant KRAS sequences were digested by RGEN with perfect match crRNA or weakened single-base mismatch crRNA. The weakened crRNA selected for genotyping is boxed at the top of the gel. [Figure 32a] Figure 32 shows the genotyping of PIK3CA point mutation (c.3140 A>G). (a) RGEN-RFLP analysis of PIK3CA mutation (c.3140 A>G) in cancer cell lines. PCR products from HeLa cells (used as wild-type control) or HCT116 cells (heterozygous for the point mutation) were digested by RGEN with perfectly matched crRNA specific for the wild-type or mutant sequence. The PIK3CA genotype in these cells was confirmed by Sanger sequencing. [Figure 32b]Figure 32 shows the genotyping of the PIK3CA point mutation (c.3140 A>G). (b) Plasmids containing either wild-type or mutant PIK3CA sequences were digested by RGEN with perfect match crRNA or weakened single-base mismatch crRNA. The weakened crRNA selected for genotyping is boxed at the top of the gel. [Figure 33a] Figure 33 shows genotyping of recurrent point mutations in cancer cell lines. (a) RGEN-RFLP assay of recurrent oncogenic point mutations in the IDH gene (c.394c>T). The genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are boxed. The black arrow indicates the DNA band cleaved by RGEN. [Figure 33b] Figure 33 shows the genotyping of recurrent point mutations in cancer cell lines. (b) RGEN-RFLP assay of recurrent oncogenic point mutations in the PIK3CA gene (c.3140A>G). The genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are boxed. The black arrow indicates the DNA band cleaved by RGEN. [Figure 33c] Figure 33 shows the genotyping of recurrent point mutations in cancer cell lines. (c) RGEN-RFLP assay of recurrent oncogenic point mutations in the NRAS gene (c.181C>A). The genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are boxed. The black arrow indicates the DNA band cleaved by RGEN. [Fig. 33d] Figure 33 shows genotyping of recurrent point mutations in cancer cell lines. (d) RGEN-RFLP assay of recurrent oncogenic point mutations in the BRAF gene (c.1799T>A). The genotypes of each cell line confirmed by Sanger sequencing are shown. Mismatched nucleotides are boxed. The black arrow indicates the DNA band cleaved by RGEN. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] According to one embodiment of the present invention, the present invention provides a composition for cleaving a target DNA in a eukaryotic cell or eukaryotic organism, comprising a guide RNA specific to the target DNA or a DNA encoding the guide RNA, and a nucleic acid encoding a Cas protein or a Cas protein. In addition, the present invention provides the use of a composition for cleaving a target DNA in a eukaryotic cell or eukaryotic organism, comprising a guide RNA specific to the target DNA or a DNA encoding the guide RNA, and a nucleic acid encoding a Cas protein or a Cas protein. In the present invention, the composition is also referred to as an RNA-guided endonuclease (RGEN) composition.

[0024] ZFN and TALEN allow targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequency obtained by each nuclease varies widely. Moreover, some ZFN and TALEN cannot show genome editing activity. DNA methylation can limit the binding of these artificial nucleases to target sites. In addition, making custom nucleases is technically difficult and time-consuming. The present inventors have overcome the shortcomings of ZFNs and TALENs by developing a new RNA-guided endonuclease composition based on Cas proteins.

[0025] Prior to the present invention, the endonuclease activity of Cas protein was known. However, due to the complexity of the genome of eukaryotic organisms, it was not known whether the endonuclease activity of Cas protein functions in eukaryotic cells. Furthermore, until now, no composition has been developed that contains Cas protein or Cas protein-encoding nucleic acid and guide RNA specific to the target DNA for cleaving target DNA in eukaryotic cells or organisms.

[0026] Compared to ZFN and TALEN, the present RGEN composition based on Cas protein can be more easily customized because new genome editing nucleases can be created by simply replacing the synthetic guide RNA component. No subcloning step is involved in the creation of customized RNA-guided endonucleases. In addition, the relatively small size of Cas genes (e.g., 4.2 kbp for Cas9) compared to a pair of TALEN genes (~6 kbp) gives the present RNA-guided endonuclease composition an advantage in some applications, such as virus-mediated gene delivery. Furthermore, the RNA-guided endonuclease does not have off-target effects, so it does not cause undesired mutations, deletions, inversions, and duplications. These properties make the present RNA-guided endonuclease composition a scalable, versatile, and convenient tool for genome engineering in eukaryotic cells and organisms. In addition, RGENs can be designed to target any DNA sequence, and almost any single nucleotide polymorphism or small insertion / deletion (indel) can be analyzed by RGEN-mediated RFLP. The specificity of RGENs is determined by the RNA component that hybridizes with target DNA sequences up to 20 base pairs (bp) in length and the Cas9 protein that recognizes the protospacer adjacent motif (PAM). RGENs are easily reprogrammed by replacing the RNA component. Thus, RGENs provide a basis for using simple and reliable RFLP analysis for various sequence mutations.

[0027] The target DNA can be endogenous DNA or artificial DNA, preferably endogenous DNA. As used herein, the term "Cas protein" refers to an essential protein component in the CRISPR / Cas system, which forms an active endonuclease or nickase when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Cas gene and protein information is available without restriction from GenBank at the National Center for Biotechnology Information (NCBI).

[0028] CRISPR-associated (cas) genes, which code for Cas proteins, are often associated with CRISPR repeat-spacer arrays. More than 40 different Cas protein families have been described. Of these protein families, Cas1 appears to be ubiquitous across various CRISPR / Cas systems. There are three types of CRISPR-Cas systems. Among them, the type II CRISPR / Cas system, which includes the Cas9 protein and the crRNA and tracrRNA, is representative and well-known. Eight CRISPR subtypes have been defined using specific combinations of cas genes and repeat structures (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube).

[0029] The Cas protein may be linked to a protein transduction domain, which may be, but is not limited to, polyarginine or the TAT protein from HIV. The composition may contain the Cas components in the form of proteins or in the form of nucleic acids encoding the Cas proteins. In the present invention, the Cas protein can be any Cas protein as long as it has endonuclease or nickase activity when complexed with a guide RNA. Preferably, the Cas protein is a Cas9 protein or a variant thereof. The variant of the Cas9 protein may be a mutant form of Cas9 in which the catalytic aspartic acid residue is changed to any other amino acid, preferably, but not limited to, alanine.

[0030] Furthermore, the Cas protein may be isolated from an organism such as, but not limited to, Streptococcus sp., preferably Streptococcus pyogenes, or may be a recombinant protein. The Cas protein from Streptococcus pyogenes can recognize the NGG trinucleotide. The Cas protein can include, but is not limited to, the amino acid sequence of SEQ ID NO: 109.

[0031] The term "recombinant," for example, when used with respect to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant Cas protein can be produced by reconstituting a Cas protein coding sequence using a human codon table.

[0032] In the present invention, the Cas protein-encoding nucleic acid may be in the form of a vector, such as a plasmid, that contains a Cas coding sequence under the control of a promoter, such as CMV or CAG. When the Cas protein is Cas9, the Cas9 coding sequence may be derived from Streptococcus sp., preferably from Streptococcus pyogenes. For example, the Cas9-encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NO: 1. Furthermore, the Cas9-encoding nucleic acid may comprise, but is not limited to, a nucleotide sequence having at least 50% homology to the sequence of SEQ ID NO: 1, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% homology to SEQ ID NO: 1. The Cas9-encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NO: 108, 110, 106, or 107.

[0033] As used herein, the term "guide RNA" refers to an RNA that is specific for a target DNA and can form a complex with a Cas protein to bring the Cas protein to the target DNA. In the present invention, the guide RNA is composed of two RNAs, namely, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or it can be a single-stranded RNA (sgRNA) made by fusion of the essential parts of crRNA and tracrRNA. The guide RNA may be a dual RNA comprising crRNA and tracrRNA. Any guide RNA can be used in the present invention, provided that the guide RNA contains the essential portions of the crRNA and tracrRNA and the portion complementary to the target.

[0034] The crRNA may hybridize to the target DNA. RGENs are composed of Cas proteins and dual RNA (invariant tracrRNA and target-specific crRNA), or Cas proteins and sgRNA (fusion of essential parts of invariant tracrRNA and target-specific crRNA), and can be easily reprogrammed by replacement of the crRNA. The guide RNA further comprises one or more additional nucleotides at the 5' end of the single-stranded guide RNA or the crRNA of the dual RNA. Preferably, the guide RNA further comprises two additional guanine nucleotides at the 5' end of the single-stranded guide RNA or the crRNA of the dual RNA.

[0035] Guide RNA can be introduced into cells or organisms in the form of RNA or DNA encoding guide RNA. Guide RNA can be in the form of isolated RNA, RNA incorporated into a viral vector, or encoded by a vector. Preferably, the vector can be, but is not limited to, a viral vector, a plasmid vector, or an Agrobacterium vector. The DNA encoding the guide RNA can be a vector that contains a sequence encoding the guide RNA. For example, the guide RNA can be transferred to a cell or organism by transfecting the cell or organism with an isolated guide RNA or a plasmid DNA that contains a sequence encoding the guide RNA and a promoter. Alternatively, the guide RNA can be introduced into a cell or organism using viral-mediated gene delivery.

[0036] When the guide RNA is transfected into a cell or organism in the form of isolated RNA, the guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably transferred into a cell in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA. As used herein, the term "isolated RNA" can be interchangeable with "naked RNA". This does not require a cloning step, which saves cost and time. However, the use of plasmid DNA or virus-mediated gene delivery for the transfection of the guide RNA is not excluded.

[0037] The RGEN composition containing a Cas protein or a Cas protein-encoding nucleic acid and a guide RNA can specifically cleave a target DNA due to the specificity of the guide RNA for the target and the endonuclease or nickase activity of the Cas protein. As used herein, the term "cleavage" refers to the cleavage of the covalent backbone of a nucleotide molecule. In the present invention, the guide RNA can be prepared to be specific for any target to be cleaved. Thus, the RGEN composition can cleave any target DNA by manipulating or genotyping the target-specific portion of the guide RNA.

[0038] Guide RNA and Cas protein can function as a pair. As used herein, the term "paired Cas nickase" can refer to guide RNA and Cas protein that function as a pair. The pair includes two guide RNAs. Guide RNA and Cas protein function as a pair to generate two nicks on different DNA strands. The two nicks can be at least 100bp apart, but are not limited thereto. In the examples, we confirmed that paired Cas nickases enable targeted mutagenesis and large deletions of chromosome segments up to 1-kbp in human cells. Importantly, paired nickases did not cause indels at off-target sites where their corresponding nucleases induce mutations. Moreover, unlike nucleases, paired nickases did not promote undesired translocations associated with off-target DNA cleavage. In principle, paired nickases double the specificity of Cas9-mediated mutagenesis, expanding the utility of RNA-guided enzymes in applications requiring precise genome editing, such as gene therapy and cell therapy.

[0039] In the present invention, the compositions may be used for genotyping the genome of eukaryotic cells or organisms in vitro. In one particular embodiment, the guide RNA may comprise the nucleotide sequence of SEQ ID NO:1, although the portion from nucleotide positions 3 to 22 thereof is a target-specific portion, and therefore the sequence of this portion may vary depending on the target. As used herein, a eukaryotic cell or eukaryote can be, but is not limited to, yeast, fungi, protists, plants, higher plants, and insect or amphibian cells, as commonly used in the art, or mammalian cells, such as CHO, HeLa, HEK293, and COS-1, including cultured cells (in vitro), transplanted and primary cultured cells (in vitro and ex vivo), and in vivo cells, and mammalian cells, such as human cells.

[0040] In one particular embodiment, it was found that the Cas9 protein / single-stranded guide RNA can generate site-specific DNA double-strand breaks in vitro and in mammalian cells, the spontaneous repair of which induces targeted genomic mutations at high frequency. Furthermore, it was found that gene knockout mice could be derived by injection of Cas9 protein / guide RNA complexes or Cas9 mRNA / guide RNA into one-cell stage embryos, and that germline-transmissible mutations could be generated by the Cas9 / guide RNA system. Using Cas proteins, rather than nucleic acids encoding Cas proteins, to induce targeted mutagenesis is advantageous because no foreign DNA is introduced into the organism. Thus, compositions containing Cas proteins and guide RNAs can be used to develop therapeutic or value-added crops, livestock, poultry, fish, pets, etc.

[0041] In another embodiment of the present invention, the present invention provides a composition for inducing targeted mutagenesis in eukaryotic cells or eukaryotic organisms, comprising a guide RNA or a DNA encoding a guide RNA specific to a target DNA, and a nucleic acid encoding a Cas protein or a Cas protein.In addition, the present invention provides the use of a composition for inducing targeted mutagenesis in eukaryotic cells or eukaryotic organisms, comprising a guide RNA or a DNA encoding a guide RNA specific to a target DNA, and a nucleic acid encoding a Cas protein or a Cas protein. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above.

[0042] In another embodiment of the present invention, the present invention provides a kit for cleaving a target DNA in a eukaryotic cell or organism, or for inducing targeted mutagenesis in a eukaryotic cell or organism, comprising a guide RNA specific for the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above. The kit may comprise a guide RNA and a Cas protein-encoding nucleic acid or a Cas protein as separate components or as one composition. The kit may include several additional components necessary for transferring the guide RNA and Cas components into a cell or organism. For example, the kit may include, but is not limited to, an injection buffer, such as a DEPC-treated injection buffer, and materials necessary for analyzing mutations in the target DNA.

[0043] In another aspect, the present invention provides a method for producing a eukaryotic cell or organism having a Cas protein and a guide RNA, comprising co-transfecting or sequentially transfecting a eukaryotic cell or organism with a Cas protein-encoding nucleic acid or Cas protein and a guide RNA or DNA encoding the guide RNA. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above.

[0044] In the present invention, Cas protein-encoding nucleic acid or Cas protein and guide RNA or DNA encoding guide RNA can be transferred into cells by various methods known in the art, such as, but not limited to, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection into protoplasts. Cas protein-encoding nucleic acid or Cas protein and guide RNA can also be transferred into organisms by various methods known in the art for administering genes or proteins, such as injection. Cas protein-encoding nucleic acid or Cas protein can be transferred into cells in the form of a complex with guide RNA or separately. Cas protein fused with a protein transduction domain, such as Tat, can also be efficiently delivered into cells. Preferably, the eukaryotic cell or organism is co-transfected or sequentially transfected with the Cas9 protein and the guide RNA. Sequential transfection can be performed by first transfection with a Cas protein-encoding nucleic acid followed by a second transfection with a naked guide RNA, preferably, but not limited to, the second transfection 3, 6, 12, 18, 24 hours later.

[0045] In another aspect, the present invention provides a eukaryotic cell or organism containing a guide RNA specific for a target DNA or a DNA encoding a guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. Eukaryotic cells or organisms can be produced by introducing into the cell or organism a composition containing a guide RNA specific to the target DNA or a DNA encoding the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein. Eukaryotic cells can be, but are not limited to, yeast, fungi, protists, higher plants, and insect or amphibian cells, as commonly used in the art, or mammalian cells, such as CHO, HeLa, HEK293, and COS-1, including cultured cells (in vitro), transplanted cells and primary cultured cells (in vitro and ex vivo), and in vivo cells, and mammalian cells, such as humans. Furthermore, the organism can be a yeast, fungus, protist, plant, higher plant, insect, amphibian, or mammal.

[0046] In another aspect of the invention, the invention provides a method for cleaving a target DNA or inducing targeted mutagenesis in a eukaryotic cell or organism, comprising treating a cell or organism having the target DNA with a composition containing a guide RNA specific for the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein. The step of treating a cell or organism with the composition may be carried out by introducing into the cell or organism the composition containing a guide RNA specific to the target DNA or a DNA encoding the guide RNA and a Cas protein-encoding nucleic acid or a Cas protein. As described above, such transfer can be performed by microinjection, transfection, electroporation, and the like.

[0047] In another embodiment of the present invention, the present invention provides an embryo having a genome edited by the present RGEN composition containing a guide RNA specific to a target DNA or a DNA encoding a guide RNA and a Cas protein-encoding nucleic acid or a Cas protein. Any embryo can be used in the present invention, and for the purposes of the present invention, the embryo can be a mouse embryo. Embryos can be produced by injecting PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin) into 4-7 week old female mice, mating the superovulated female mice with males, and collecting the fertilized embryos from the oviducts. The RGEN composition introduced into the embryo can cleave the target DNA complementary to the guide RNA through the action of the Cas protein, causing a mutation in the target DNA. Thus, the embryo into which the RGEN composition is introduced has an edited genome.

[0048] In one particular embodiment, it has been found that the RGEN composition can induce mutations in mouse embryos that can be transmitted to offspring. The method of introducing the RGEN composition into the embryo can be any method known in the art, such as microinjection, stem cell insertion, retrovirus insertion, etc. Preferably, the microinjection method can be used.

[0049] In another aspect, the present invention provides a genome-modified animal obtained by transferring an embryo having a genome edited by the RGEN composition into the oviduct of an animal. In the present invention, the term "genome-modified animal" refers to an animal whose genome has been modified by the RGEN composition at the embryonic stage, and the type of the animal is not limited. The genome modified animal has a mutation caused by targeted mutagenesis based on the RGEN composition. The mutation can be any one of deletion, insertion, translocation, and inversion. The site of the mutation is determined by the sequence of the guide RNA of the RGEN composition. Genomically modified animals carrying a gene mutation can be used to determine the function of that gene.

[0050] In another aspect of the present invention, the present invention provides a method for producing a genome-modified animal, comprising introducing the RGEN composition containing a guide RNA or DNA encoding a guide RNA specific for a target DNA and a Cas protein-encoding nucleic acid or Cas protein into an animal embryo; and transferring the embryo into the oviduct of a pseudopregnant surrogate mother to produce a genome-modified animal. The step of introducing the RGEN composition can be accomplished by any method known in the art, such as microinjection, stem cell insertion, retroviral insertion, etc.

[0051] In another aspect of the present invention, the present invention provides plants regenerated from genome-modified protoplasts prepared by a method for eukaryotic cells comprising an RGEN composition. In another embodiment of the present invention, the present invention provides a composition for genotyping mutations or polymorphisms in an isolated biological sample, comprising a guide RNA and a Cas protein specific to a target DNA sequence.In addition, the present invention provides a composition for genotyping the nucleic acid sequence of a pathogenic microorganism in an isolated biological sample, comprising a guide RNA and a Cas protein specific to a target DNA sequence. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above.

[0052] As used herein, the term "genotyping" refers to a "restriction fragment length polymorphism (RFLP) assay." RFLPs can be used to 1) detect indels in cells or organisms induced by artificial nucleases, 2) genotype naturally occurring mutations or polymorphisms in cells or organisms, or 3) genotype the DNA of infectious pathogenic microorganisms, including viruses or bacteria. Mutations or polymorphisms can be induced in cells by artificial nucleases. The artificial nuclease may be, but is not limited to, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RGEN. As used herein, the term "biological sample" includes, but is not limited to, a sample for analysis, such as tissue, cells, whole blood, serum (semm), plasma, saliva, sputum, cerbrospinal fluid, or urine.

[0053] The mutation or polymorphism may be a naturally occurring mutation or polymorphism. The mutation or polymorphism is induced by a pathogenic microorganism, i.e., the mutation or polymorphism occurs due to infection with a pathogenic microorganism if a pathogenic microorganism is detected and the biological sample is confirmed as infected. The pathogenic microorganism may be, but is not limited to, a virus or a bacterium.

[0054] Artificial nuclease-induced mutations are detected by a variety of methods, including mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescent PCR, DNA melting analysis, as well as Sanger and deep sequencing. Although the T7E1 and Surveyor assays are widely used, these assays detect heteroduplexes (formed by hybridization of a mutant sequence with a wild-type sequence or two different mutant sequences); they cannot detect homoduplexes formed by hybridization of two identical mutant sequences, and therefore often underestimate mutation frequencies. Thus, these assays cannot distinguish between homozygous biallelic mutant clones and wild-type cells, and cannot distinguish between heterozygous biallelic mutants and heterozygous monoallelic mutants (Figure 22). In addition, sequence polymorphisms near the nuclease target site can produce confounding results since the enzyme can cleave the heteroduplex formed by the hybridization of these different wild-type alleles. RFLP analysis is the preferred method since it does not have these constraints. In fact, RFLP analysis was one of the first methods used to detect artificial nuclease-mediated mutations. Unfortunately, however, it is limited by the availability of suitable restriction enzyme recognition sites.

[0055] In another embodiment of the present invention, the present invention provides a kit for genotyping mutations or polymorphisms in an isolated biological sample, comprising a composition for genotyping mutations or polymorphisms in an isolated biological sample.In addition, the present invention provides a kit for genotyping the nucleic acid sequence of a pathogenic microorganism in an isolated biological sample, comprising a guide RNA specific to a target DNA sequence and a Cas protein. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above.

[0056] In another aspect of the present invention, the present invention provides a method for genotyping mutations or polymorphisms in an isolated biological sample, using a composition for genotyping mutations or polymorphisms in an isolated biological sample.In addition, the present invention provides a method for genotyping a nucleic acid sequence of a pathogenic microorganism in an isolated biological sample, comprising a guide RNA and a Cas protein specific to a target DNA sequence. The guide RNA, the Cas protein-encoding nucleic acid or the Cas protein are as described above.

[0057] (Example) In the following, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples. EXAMPLES

[0058] Genome editing assays 1-1. DNA cleavage activity of Cas9 protein First, the DNA cleavage activity of Cas9 from Streptococcus pyogenes was tested in vitro in the presence or absence of chimeric guide RNAs. For this purpose, recombinant Cas9 protein expressed and purified in E. coli was used to cleave predigested or circular plasmid DNA containing the 23-base pair (bp) human CCR5 target sequence, which consists of a 20-bp DNA sequence complementary to the crRNA or chimeric guide RNA and a trinucleotide (5'-NGG-3') protospacer adjacent motif (PAM) recognized by Cas9 itself (Figure 1a).

[0059] Specifically, the Cas9 coding sequence (4,104 bp) from Streptococcus pyogenes strain M1 GAS (NC_002737.1) was reconstructed using a human codon usage table and synthesized using oligonucleotides. First, 1-kb DNA segments were assembled using overlapping 35-mer oligonucleotides and Phusion polymerase (New England Biolabs) and cloned into a T-vector (SolGent). The full-length Cas9 sequence was assembled by overlapping PCR using four 1-kbp DNA segments. The Cas9 coding DNA segment was subcloned into p3s derived from pcDNA3.1 (Invitrogen). In this vector, a peptide tag (NH2-GGSGPPKKKRKVYPYDVPDYA-COOH, SEQ ID NO:2) containing an HA epitope and a nuclear localization signal (NLS) was added to the C-terminus of Cas9. Expression and nuclear localization of Cas9 protein in HEK 293T cells was confirmed by Western blotting using an anti-HA antibody (Santa Cruz).

[0060] The Cas9 cassette was then subcloned into pET28-b(+) and transformed into BL21(DE3). Expression of Cas9 was induced with 0.5 mM IPTG for 4 h at 25°C. Cas9 protein with a C-terminal His6-tag was purified using Ni-NTA agarose resin (Qiagen) and dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol (1). Purified Cas9 (50 nM) was incubated with supercoiled plasmid DNA or predigested plasmid DNA (300 ng) and chimeric RNA (50 nM) in a reaction volume of 20 μl in NEB buffer 3 at 37°C for 1 h. Digested DNA was analyzed by electrophoresis using a 0.8% agarose gel. Cas9 efficiently cleaved plasmid DNA at the predicted location only in the presence of synthetic RNA and did not cleave a control plasmid lacking the target sequence (Figure 1b).

[0061] 1-2. DNA cleavage by Cas9 / guide RNA complex in human cells Using an RFP-GFP reporter, we investigated whether the Cas9 / guide RNA complex could cleave a target sequence integrated between the RFP and GFP sequences in mammalian cells. In this reporter, a GFP sequence is frameshifted and fused to an RFP sequence.(2) Active GFP is expressed only when the target sequence is cleaved by a site-specific nuclease and non-homologous end joining (NHEJ) repair of the error-prone double-strand break (DSB) generates a frameshifted small insertion or deletion (indel) near the target sequence (Fig. 2).

[0062] The RFP-GFP reporter plasmid used in this study was constructed as previously described (2). Oligonucleotides (Table 1) corresponding to the target sites were synthesized (Macrogen) and annealed. The annealed oligonucleotides were ligated into the reporter vector digested with EcoRI and BamHI. HEK 293T cells were co-transfected with Cas9-encoding plasmid (0.8 μg) and RFP-GFP reporter plasmid (0.2 μg) in 24-well plates using Lipofectamine 2000 (Invitrogen).

[0063] Meanwhile, in vitro transcribed chimeric RNA was prepared as follows. RNA was in vitro transcribed using MEGAshortscript T7 kit (Ambion) by run-off reaction according to the manufacturer's instructions. Templates for in vitro transcription of RNA were generated by annealing of two complementary single-stranded DNAs or by PCR amplification (Table 1). Transcribed RNA was separated on an 8% denaturing urea-PAGE gel. The gel slice containing RNA was excised and transferred to probe elution buffer. RNA was recovered in nuclease-free water, followed by phenol-chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry.

[0064] Twelve hours after transfection, chimeric RNA (1 μg) prepared by in vitro transcription was transfected using Lipofectamine 2000. Three days after transfection, the transfected cells were subjected to flow cytometry to count cells expressing both RFP and GFP. It was found that GFP-expressing cells were obtained only when the cells were first transfected with the Cas9 plasmid and then with the guide RNA 12 hours later (Figure 2), demonstrating that RGEN can recognize and cleave target DNA sequences in cultured human cells. Thus, GFP-expressing cells were obtained by sequential transfection of the Cas9 plasmid and the guide RNA rather than by co-transfection. [Table 1]

[0065] 1-3. Targeted destruction of endogenous genes in mammalian cells by RGEN To test whether RGEN can be used for targeted disruption of endogenous genes in mammalian cells, genomic DNA isolated from transfected cells was analyzed using T7 endonuclease I (T7E1), a mismatch-sensitive endonuclease that specifically recognizes and cleaves heteroduplexes formed by hybridization of wild-type and mutant DNA sequences (3). To introduce DSBs into mammalian cells using RGEN, 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) was used according to the manufacturer's protocol, and 2 × 10 6 K562 cells were transfected with 20 μg of Cas9-encoding plasmid. For this experiment, K562 (ATCC, CCL-243) cells were grown in RPMI-1640 containing 10% FBS and a penicillin / streptomycin mixture (100 U / ml and 100 μg / ml, respectively).

[0066] After 24 h, 10–40 μg of in vitro transcribed chimeric RNA was added to 1 × 10 6 K562 cells were nucleofected. In vitro transcribed chimeric RNA was prepared as described in Example 1-2. Two days after RNA transfection, cells were harvested and genomic DNA was isolated. Regions containing the target sites were PCR amplified using the primers listed in Table 1. Amplicons were subjected to T7E1 assay as previously described (3). For sequence analysis, PCR products corresponding to genomic modifications were purified and cloned into T-Blunt vectors using the T-Blunt PCR Cloning Kit (SolGent). Cloned products were sequenced using M13 primers.

[0067] Mutations were found to be induced only when cells were transfected sequentially with a Cas9-encoding plasmid followed by a guide RNA (Figure 3). The mutation frequency estimated from the relative DNA band intensities (Indels (%) in Figure 3a) was RNA dose-dependent and ranged from 1.3% to 5.1%. DNA sequence analysis of PCR amplicons confirmed the induction of RGEN-mediated mutations at endogenous sites. Indels and microhomologies, hallmarks of error-prone NHEJ, were observed at the target site. The mutation frequency measured by direct sequencing was 7.3% (=7 mutant clones / 96 clones), comparable to that obtained by zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs). To induce mutations in cells, sequential transfection of the Cas9 plasmid and the guide RNA was required, but in the case of the guide RNA-encoding plasmid, sequential transfection was not necessary and cells were co-transfected with the Cas9 plasmid and the guide RNA-encoding plasmid.

[0068] Meanwhile, both ZFNs and TALENs have been successfully developed to disrupt the human CCR5 gene, which encodes a G protein-coupled chemokine receptor that is an essential coreceptor for HIV infection (3-6). CCR5-specific ZFNs are currently in clinical trials for AIDS treatment in the United States (7). However, these ZFNs and TALENs have off-target effects, including both local mutations at sites whose sequences are homologous to the on-target sequence (6, 8-10), as well as genomic rearrangements resulting from the repair of two simultaneous DSBs induced at the on-target and off-target sites (11-12). The most prominent off-target site associated with these CCR5-specific artificial nucleases is present in the CCR2 locus, a close homologue of CCR5, located 15-kbp upstream of CCR5. To avoid off-target mutations in the CCR2 gene and undesirable deletions, inversions, and duplications of the 15-kbp chromosomal segment between the CCR5 on-target site and the CCR2 off-target site, we purposely selected the target site of our CCR5-specific RGEN to recognize a region in the CCR5 sequence that has no obvious homology with the CCR2 sequence.

[0069] We investigated whether CCR5-specific RGEN has off-target effects. To this end, we searched for potential off-target sites in the human genome by identifying sites that are approximately homologous to the 23-bp target sequence of interest. As expected, no such sites were found in the CCR2 gene. Instead, four sites were found, each with a three-base mismatch with the on-target site (Fig. 4a). The T7E1 assay showed that no mutations were detected at these sites (assay sensitivity, ~0.5%), demonstrating the excellent specificity of RGEN (Fig. 4b). Furthermore, PCR was used to detect the induction of chromosomal deletions in cells transfected separately with CCR5-specific ZFN and RGEN-encoding plasmids. ZFN induced deletions, but RGEN did not (Fig. 4c).

[0070] Next, we reprogrammed RGEN by replacing the CCR5-specific guide RNA with a new synthetic RNA designed to target the human C4BPB gene, which encodes the β-chain of the transcription factor C4b-binding protein. This RGEN induced high frequency mutations at the chromosomal target site in K562 cells (Figure 3b). The mutation frequencies measured by the T7E1 assay and direct sequencing were 14% and 8.3% (=4 mutant clones / 48 clones), respectively. Of the four mutant sequences, two clones contained one- or two-base insertions exactly at the cleavage site, a pattern also observed at the CCR5 target site. These results indicate that RGEN cleaves the chromosomal target DNA at the expected location in the cell. EXAMPLES

[0071] Genome editing mediated by protein-based RGEN RGEN can be delivered into cells in many different forms. RGEN is composed of Cas9 protein, crRNA, and tracrRNA. The two RNAs can be fused to form a single-stranded guide RNA (sgRNA). A plasmid encoding Cas9 under the control of a promoter such as CMV or CAG can be transfected into cells. crRNA, tracrRNA, or sgRNA can also be expressed in cells using plasmids encoding these RNAs. However, the use of plasmids often leads to integration of the whole or part of the plasmid into the host genome. Bacterial sequences incorporated into the plasmid DNA can cause unwanted immune responses in vivo. Animals and plants derived from plasmid-transfected or DNA-transfected cells for cell therapy must go through costly and lengthy regulatory procedures before marketing approval in most developed countries. Furthermore, plasmid DNA can remain in cells for several days after transfection, exacerbating the off-target effects of RGEN.

[0072] Here, we used recombinant Cas9 protein complexed with in vitro transcribed guide RNA to induce targeted disruption of endogenous genes in human cells. Recombinant Cas9 protein fused with a hexahistidine tag was expressed in E. coli and purified using standard Ni-ion affinity chromatography and gel filtration. Purified recombinant Cas9 protein was concentrated in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol). Cas9 protein / sgRNA complex was directly introduced into K562 cells by nucleofection. Specifically, 1 × 10 22.5–225 (1.4–14 μM) Cas9 protein mixed with 100 μg (29 μM) of in vitro transcribed sgRNA (or 40 μg crRNA and 80 μg tracrRNA) in 100 μl of solution was used with the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. 6 K562 cells were transfected. After nucleofection, cells were placed in growth medium in 6-well plates and incubated for 48 hours. 2 × 10 5 K562 cells were transfected in a 5-fold scaled-down manner using 4.5–45 μg of Cas9 protein mixed with 6–60 μg of in vitro transcribed sgRNA (or 8 μg of crRNA and 16 μg of tracrRNA) in 20 μl of solution. Nucleofected cells were then placed in growth medium in 48-well plates. After 48 h, cells were harvested and genomic DNA was isolated. Genomic DNA regions spanning the target sites were PCR amplified and subjected to the T7E1 assay.

[0073] As shown in Figure 10, the Cas9 protein / sgRNA complex induced targeted mutations in the CCR5 locus at frequencies of 4.8–38% in a dose-dependent manner of sgRNA or Cas9 protein, which was similar to the frequency obtained by transfection of Cas9 plasmid (45%). The Cas9 protein / crRNA / tracrRNA complex was able to induce mutations at a frequency of 9.4%. The Cas9 protein alone was unable to induce mutations. 2 × 10 5 When cells were transfected with 5-fold scaled-down doses of Cas9 protein and sgRNA, mutation frequencies at the CCR5 locus ranged from 2.7 to 57% in a dose-dependent manner, which was higher than the frequency obtained by cotransfection of Cas9 and sgRNA plasmids (32%).

[0074] We also tested a Cas9 protein / sgRNA complex targeting the ABCC11 gene and found that this complex induced indels at a frequency of 35%, demonstrating the versatility of this method. [Table 2] EXAMPLES

[0075] RNA-guided genome editing in mice To investigate the gene targeting capability of RGEN in mouse embryos at the pronuclear (PN) stage, the forkhead box N1 (Foxn1) gene, which is important for thymus development and keratinocyte differentiation (Nehls et al., 1996), and the protein kinase, DNA activated, catalytic polypeptide (Prkdc) gene, which encodes an enzyme important for DNA DSB repair and recombination (Taccioli et al., 1998), were used. To evaluate the genome editing activity of Foxn1-RGEN, we injected Cas9 mRNA (10-ng / μl solution) together with various doses of sgRNA into the cytoplasm of mouse embryos at the PN stage (Fig. 5a) and performed a T7 endonuclease I (T7E1) assay (Kim et al. 2009) using genomic DNA obtained from in vitro cultured embryos (Fig. 6a).

[0076] Alternatively, we directly injected RGEN into the cytoplasm or pronucleus of one-cell mouse embryos in the form of recombinant Cas9 protein (0.3–30 ng / μl) complexed with a two-fold molar excess of Foxn1-specific sgRNA (0.14–14 ng / μl) and analyzed Foxn1 gene mutations using in vitro cultured embryos (Figure 7). Specifically, Cas9 mRNA and sgRNA were synthesized in vitro from linear DNA templates using mMESSAGE mMACHINE T7 Ultra kit (Ambion) and MEGAshortscript T7 kit (Ambion), respectively, according to the manufacturer's instructions, and diluted with an appropriate amount of diethylpyrocarbonate (DEPC, Sigma)-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4). Templates for sgRNA synthesis were generated using the oligonucleotides listed in Table 3. Recombinant Cas9 protein was obtained from ToolGen, Inc. [Table 3]

[0077] All animal experiments were performed in accordance with the Korea Food and Drug Administration (KFDA) guidelines. The protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Facility of Yonsei University (Permit Number: 2013-0099). All mice were maintained in a specific pathogen-free facility at the Yonsei Laboratory Animal Research Facility. FVB / NTac (Taconic) and ICR mouse strains were used as embryo donors and surrogate mothers, respectively. Female FVB / NTac mice (7–8 weeks old) were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG, Sigma) and 5 IU human chorionic gonadotropin (hCG, Sigma) at 48-h intervals. Superovulated female mice were mated with FVB / NTac stud males, and fertilized embryos were collected from the oviducts. Using a Piezo-driven micromanipulator (Prime Tech), Cas9 mRNA and sgRNA in M2 medium (Sigma) were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei.

[0078] For recombinant Cas9 protein injection, the recombinant Cas9 protein:Foxn1-sgRNA complex was diluted in DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4) and injected into the male pronucleus using a TransferMan NK2 micromanipulator and a FemtoJet microinjector (Eppendorf). The manipulated embryos were transferred into the oviducts of pseudopregnant surrogate mothers to generate live animals or cultured in vitro for further analysis.

[0079] To screen F0 mice and in vitro cultured mouse embryos for RGEN-induced mutations, T7E1 assays were performed using genomic DNA samples from tail biopsies and whole embryo lysates as previously described ( Cho et al., 2013 ). Briefly, the genomic region containing the RGEN target site was PCR amplified, melted, and reannealed to form heteroduplex DNA, which was treated with T7 endonuclease 1 (New England Biolabs) and then analyzed by agarose gel electrophoresis. Potential off-target sites were identified by a search using bowtie 0.12.9, which was also examined by the T7E1 assay. The primer pairs used in these assays are listed in Tables 4 and 5. [Table 4] [Table 5]

[0080] Mutant founders identified by the T7E1 assay were further analyzed by fPCR. Appropriate regions of genomic DNA were sequenced as previously described (Sung et al., 2013). For routine PCR genotyping of F1 progeny, the following primer pairs were used for both wild-type and mutant alleles: 5'-CTACTCCCTCCGCAGTCTGA-3' (SEQ ID NO: 69) and 5'-CCAGGCCTAGGTTCCAGGTA-3' (SEQ ID NO: 70) for the Foxn1 gene, and 5'-CCCCAGCATTGCAGATTTCC-3' (SEQ ID NO: 71) and 5'-AGGGCTTCTTCTCTACAATCACG-3' (SEQ ID NO: 72) for the Prkdc gene.

[0081] In the case of Cas9 mRNA injection, the percentage of mutants (number of mutant embryos / number of all embryos) was dose-dependent, ranging from 33% (1 ng / μl sgRNA) to 91% (100 ng / μl) (Figure 6b). Sequence analysis confirmed mutations in the Foxn1 gene, although most mutations were small deletions (Figure 6c), similar to those induced by ZFNs and TALENs (Kim et al., 2013). In the case of Cas9 protein injection, these injection doses and injection methods did not significantly affect the survival and development of mouse embryos in vitro, and more than 70% of RGEN-injected embryos hatched normally in both experiments. In addition, the percentage of mutants obtained by Cas9 protein injection was dose-dependent, reaching 88% at the highest dose by pronuclear injection and 71% by intracytoplasmic injection (Figures 7a and 7b). Similar to the mutation patterns induced by Cas9 mRNA and sgRNA (Figure 6c), the mutations induced by Cas9 protein-sgRNA complexes were mainly small deletions (Figure 7c). These results clearly demonstrate that RGEN has high gene targeting activity in mouse embryos.

[0082] Encouraged by the high mutation frequency and low cytotoxicity induced by RGEN, we generated live animals by transferring mouse embryos into the oviducts of pseudopregnant surrogate mothers. Notably, birth rates were very high, ranging from 58% to 73%, and were not affected by increasing doses of Foxn1-sgRNA (Table 6). [Table 6]

[0083] Of 147 newborns, we obtained 99 mutant founder mice. Consistent with the results observed in cultured embryos (Fig. 6b), the mutant fraction was proportional to the Foxn1-sgRNA dose, reaching a maximum of 93% (100 ng / μl Foxn1-sgRNA) (Tables 6 and 7, Fig. 5b). [Table 7] TIFF2025072369000009.tif251148 TIFF2025072369000010.tif72151

[0084] To generate Prkdc-targeted mice, we applied a 5-fold higher concentration of Cas9 mRNA (50 ng / μl) along with increasing doses of Prkdc-sgRNA (50, 100, and 250 ng / μl). Again, the birth rates were very high, ranging from 51% to 60%, resulting in a sufficient number of newborns for analysis (Table 6). The mutant percentage was 57% (21 mutant founders out of 37 newborns) at the highest dose of Prkdc-sgRNA. These birth rates obtained by RGEN were approximately 2-10 times higher than the TALEN-induced birth rates reported in our previous study (Sung et al., 2013). These results indicate that RGEN is a potent gene targeting reagent with minimal toxicity.

[0085] To test for germline transmission of the mutant allele, we crossed Foxn1 mutant founder #108, which is a mosaic with four different alleles (Figure 5c and Table 8), with wild-type mice and observed the genotype of the F1 offspring. [Table 8] TIFF2025072369000012.tif54161

[0086] As expected, all offspring were heterozygous mutants with a wild-type allele and one of the mutant alleles (Figure 5d). We also confirmed germline transmission in independent founder mice of Foxn1 (Figure 8) and Prkdc (Figure 9). To our knowledge, these results provide the first evidence that RGEN-induced mutant alleles are stably transmitted to the F1 offspring of animals. EXAMPLES

[0087] RNA-guided genome editing in plants 4-1. Production of Cas9 protein The Cas9 coding sequence (4,104 bp) from Streptococcus pyogenes strain M1 GAS (NC_002737.1) was cloned into the pET28-b(+) plasmid. A nuclear targeting sequence (NLS) was included at the N-terminus of the protein to ensure nuclear localization of the protein. The pET28-b(+) plasmid containing the Cas9 ORF was transformed into BL21(DE3). Cas9 was then induced with 0.2 mM IPTG for 16 h at 18 °C and purified using Ni-NTA agarose beads (Qiagen) according to the manufacturer's instructions. The purified Cas9 protein was concentrated using Ultracel-100K (Millipore).

[0088] 4-2. Production of guide RNA The genomic sequence of the Arabidopsis gene encoding BRI1 was screened for the presence of an NGG motif in an exon, a so-called protospacer adjacent motif (PAM), required for Cas9 targeting. To disrupt the Arabidopsis BRI1 gene, we identified two RGEN target sites in exons containing NGG motifs. sgRNAs were generated in vitro using template DNA. Each template DNA was generated by an extension reaction using two partially overlapping oligonucleotides (Macrogen, Table X1) and Phusion polymerase (Thermo Scientific) under the following conditions: 98°C 30 s (98°C 10 s, 54°C 20 s, 72°C 2 min) x 20, 72°C 5 min. [Table 9]

[0089] The extended DNA was purified and used as a template for in vitro production of guide RNA using MEGAshortscript T7 kit (Life Technologies). The guide RNA was then purified by phenol / chloroform extraction and ethanol precipitation. To prepare the Cas9 / sgRNA complex, 10 μl of purified Cas9 protein (12 μg / μl) and 4 μl each of the two sgRNAs (11 μg / μl) were mixed in 20 μl NEB3 buffer (New England Biolabs) and incubated at 37 °C for 10 min.

[0090] 4-3. Transfection of Cas9 / sgRNA complex into protoplasts Leaves from 4-week-old Arabidopsis seedlings, axenically grown in Petri dishes, were digested in enzyme solution (1% Cellulase R10, 0.5% Macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7 and CPW salts) for 8–16 h at 25 °C with shaking at 40 rpm in the dark. The enzyme / protoplast solution was filtered and centrifuged at 100 × g for 3–5 min. After counting the cells using a hemocytometer under a microscope (×100), the protoplasts were resuspended in CPW solution. Finally, the protoplasts were diluted to 1 × 10 in MMG solution (4 mM HEPES pH 5.7, 400 mM mannitol and 15 mM MgCl2). 6The protoplasts were resuspended at 1000μl / ml. To transfect the protoplasts with the Cas9 / sgRNA complex, 200μl (200,000 protoplasts) of the protoplast suspension was gently mixed with 3.3 or 10μl of the Cas9 / sgRNA complex [Cas9 protein (6μg / μl) and two sgRNAs (2.2μg / μl each)] and 200μl of 40% polyethylene glycol transfection buffer (40% PEG4000, 200mM mannitol and 100mM CaCl2) in a 2ml tube. After 5-20 min incubation at room temperature, the transfection was stopped by adding a washing buffer containing W5 solution (2mM MES pH 5.7, 154mM NaCl, 125mM CaCl2 and 5mM KCl). The protoplasts were then harvested by centrifugation at 100 × g for 5 min, washed with 1 ml of W5 solution, and centrifuged again at 100 × g for 5 min. The density of protoplasts was 1 × 10 5 / ml and cultured them in modified KM 8p liquid medium containing 400 mM glucose.

[0091] 4-4. Detection of mutations in Arabidopsis protoplasts and plants 24 or 72 hours after transfection, protoplasts were harvested and genomic DNA was isolated. The genomic DNA region spanning the two target sites was PCR amplified and subjected to T7E1 assay. As shown in FIG. 11, indels were induced by RGEN at a high frequency ranging from 50% to 70%. Unexpectedly, mutations were induced 24 hours after transfection. The Cas9 protein appears to function immediately after transfection. The PCR products were purified and cloned into the T-Blunt PCR Cloning Kit (Solgent). The plasmids were purified and subjected to Sanger sequencing using M13F primers. One mutant sequence had a 7-bp deletion at one site (FIG. 12). The other three mutant sequences had deletions of ~220-bp DNA fragments between the two RGEN sites. EXAMPLES

[0092] Cas9 protein transduction using cell-penetrating peptides or protein transduction domains 5-1. Construction of His-Cas9-encoding plasmid Cas9 with a C-terminal cysteine ​​was prepared by PCR amplification using a previously described Cas9 plasmid (Cho, 2013 #166) as a template and cloned into the pET28-(a) vector (Novagen, Merck Millipore, Germany) containing an N-terminal His tag.

[0093] 5-2. Cell culture 293T (human embryonic kidney cell line) and HeLa (human ovarian cancer cell line) were cultured in DMEM (GIBCO-BRL Rockville) supplemented with 10% FBS and 1% penicillin and streptomycin.

[0094] 5-3. Expression and purification of Cas9 protein To express Cas9 protein, E. coli BL21 cells were transformed with the pET28-(a) vector encoding Cas9 and inoculated onto Luria-Bertani (LB) agar medium containing 50 μg / mL kanamycin (Amresco, Solon, OH). The next day, a single colony was picked and grown overnight at 37 °C in LB broth containing 50 μg / mL kanamycin. The next day, this seed culture was inoculated at 0.1 OD600 into Luria broth containing 50 μg / mL kanamycin and incubated at 37 °C for 2 h until the OD600 reached 0.6-0.8. To induce Cas9 protein expression, isopropyl-β-D-thiogalactopyranoside (IPTG) (Promega, Madison, WI) was added to a final concentration of 0.5 mM, after which the cells were grown overnight at 30 °C.

[0095] Cells were harvested by centrifugation at 4000 rpm for 15–20 min, resuspended in lysis buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole, 1× protease inhibitor cocktail, 1 mg / ml lysozyme) and lysed by sonication (40% duty, 10 s pulse, 30 s pause, 10 min on ice). The soluble fraction was isolated as the supernatant after centrifugation at 15,000 rpm for 20 min at 4°C. Cas9 protein was purified at 4°C using a column containing Ni-NTA agarose resin (QIAGEN) and an AKTA prime instrument (AKTA prime, GE Healthcare, UK). During this chromatography, the soluble protein fraction was loaded onto the Ni-NTA agarose resin column (GE Healthcare, UK) at a flow rate of 1 mL / min. The column was washed with wash buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole, 1x protease inhibitor cocktail) and bound proteins were eluted with elution buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 250 mM imidazole, 1x protease inhibitor cocktail) at a flow rate of 0.5 ml / min. The pooled elution fractions were concentrated and dialyzed against storage buffer (50 mM Tris-HCl, pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol). Protein concentration was quantified by Bradford assay (Biorad, Hercules, CA) and purity was analyzed by SDS-PAGE with bovine serum albumin as a control.

[0096] 5-4. Conjugation of Cas9 with 9R4L 1 mg of Cas9 protein diluted to a concentration of 1 mg / mL in PBS and 50 μg of maleimide-9R4L peptide (Peptron, Korea) in 25 μL DW were mixed gently on a rotor for 2 h at room temperature and overnight at 4° C. To remove unconjugated maleimide-9R4L, the sample was dialyzed against DPBS (pH 7.4) for 24 h at 4° C. using a 50 kDa molecular weight cutoff membrane. Cas9-9R4L protein was recovered from the dialysis membrane and the protein amount was measured using the Bradford assay.

[0097] 5-5. Preparation of sgRNA-9R4L sgRNA (1 μg) was gently added to various amounts of C9R4LC peptide (spanning a weight ratio of 1–40) in 100 μl of DPBS (pH 7.4). The mixture was incubated at room temperature for 30 min and diluted 10-fold with RNAse-free deionized water. The hydrodynamic diameter and zeta potential of the formed nanoparticles were measured using dynamic light scattering (Zetasizer-nano analyzer ZS; Malvern instruments, Worcestershire, UK).

[0098] 5-6. Cas9 protein and sgRNA treatment Cas9-9R4L and sgRNA-C9R4LC were treated on cells as follows: 1 μg sgRNA and 15 μg C9R4LC peptide were added to 250 mL OPTIMEM medium and incubated at room temperature for 30 min. 24 h after seeding, cells were washed with OPTIMEM medium and treated with sgRNA-C9R4LC complex for 4 h at 37°C. Cells were washed again with OPTIMEM medium and treated with Cas9-9R4L for 2 h at 37°C. After treatment, the medium was replaced with serum-containing complete medium and incubated at 37°C for 24 h before the next treatment. The same procedure was performed on three consecutive days for multiple treatments of Cas9 and sgRNA.

[0099] 5-7. Cas9-9R4L and sgRNA-9R4L can edit endogenous genes in cultured mammalian cells without the use of additional delivery vehicles To determine whether Cas9-9R4L and sgRNA-9R4L can edit endogenous genes in cultured mammalian cells without the use of additional delivery means, we treated 293 cells with Cas9-9R4L and sgRNA-9R4L targeting the CCR5 gene and analyzed their genomic DNA. T7E1 assay showed that 9% of the CCR5 gene was disrupted in cells treated with both Cas9-9R4L and sgRNA-9R4L, and no CCR5 gene disruption was observed in control cells, including those that were untreated, or treated with either Cas9-9R or sgRNA-9R4L, or treated with both unmodified Cas-9 and sgRNA (Figure 13), suggesting that treatment with Cas9-9R4L protein and sgRNA conjugated with 9R4L, but not unmodified Cas-9 and sgRNA, can cause efficient genome editing in mammalian cells. EXAMPLES

[0100] Control of off-target mutations by guide RNA structure Recently, three groups reported that RGENs have off-target effects in human cells. Surprisingly, RGENs efficiently induced mutations at off-target sites that differed from the on-target site by 3 to 5 nucleotides. However, we noticed some differences between our RGENs and the RGENs used by other groups. First, we used a dual RNA of crRNA and tracrRNA rather than a single-stranded guide RNA (sgRNA) composed of the essential parts of crRNA and tracrRNA. Second, we transfected K562 cells (but not HeLa cells) with a synthetic crRNA rather than a plasmid encoding crRNA. HeLa cells were transfected with a crRNA-encoding plasmid. The other groups used an sgRNA-encoding plasmid. Third, our guide RNA has two additional guanine nucleotides at the 5' end, which are required for efficient transcription in vitro by T7 polymerase. Such additional nucleotides were not included in the sgRNA used by the other groups. Thus, the RNA sequence of our guide RNA was 5'-GGX 20 whereas 5'-GX 19 represents sequences used by other groups, and X 20 or GX 19corresponds to the 20-bp target sequence. The first guanine nucleotide is required for transcription by RNA polymerase in cells. To test whether off-target RGEN effects could be due to these differences, we selected four RGENs that induce off-target mutations at high frequencies in human cells (13). First, we compared our method using in vitro transcribed dual RNAs with the method of transfecting sgRNA-encoding plasmids in K562 cells to measure the mutation frequency at on-target and off-target sites by T7E1 assay. The three RGENs showed comparable mutation frequencies at on-target and off-target sites regardless of the guide RNA configuration. Interestingly, one RGEN (VEFGA site 1) did not induce indels at one valid off-target site that differs from the on-target site by three nucleotides (Figure 14, called OT1-11) when using synthetic dual RNA. However, synthetic dual RNA did not identify the other valid off-target site (OT1-3) that differs by two nucleotides from the on-target site.

[0101] Next, we investigated whether the addition of two guanine nucleotides to the 5' end of the sgRNA could make the RGEN more specific. 20 (or 5'-GGGX 19 ) sgRNA and 5'-GX 19 The four GXs complexed with Cas9 were tested in comparison with sgRNA. 19 sgRNAs induced indels equally efficiently at on- and off-target sites and tolerated up to four-nucleotide mismatches. In stark contrast, GGX 20 The sgRNA effectively identified off-target sites. Indeed, we 20 When using sgRNA, the T7E1 assay detected few RGEN-induced indels at six of the seven validated off-target sites (Figure 15). However, we did detect 2 GGX 20GX corresponding to sgRNA (VEGFA sites 1 and 3) 19 We noticed that sgRNAs were less active at the on-target site than sgRNAs. These results indicate that additional nucleotides at the 5' end can affect mutation frequencies at on- and off-target sites, possibly by altering the stability, concentration, or secondary structure of the guide RNA. These results were due to three factors: the use of synthetic guide RNA instead of a guide RNA-encoding plasmid, the use of dual RNA instead of sgRNA, and the use of GX 19 GGX instead of sgRNA 20 This suggests that the use of sgRNAs has a cumulative effect on identifying off-target sites. EXAMPLES

[0102] Paired Cas9 Nickase In principle, single-strand breaks (SSBs) cannot be repaired by error-prone NHEJ but trigger high-fidelity homology-directed repair (HDR) or base excision repair. However, nickase-induced targeted mutagenesis by HDR is much less efficient than nuclease-induced mutagenesis. We reasoned that paired Cas9 nickases would generate multiple DSBs that trigger DNA repair by NHEJ or HDR, resulting in efficient mutagenesis (Fig. 16a). Furthermore, paired nickases would double the specificity of Cas9 genome editing.

[0103] We first tested several Cas9 nucleases and nickases designed against a target site in the AAVS1 locus (Fig. S16b) by fluorescent capillary electrophoresis in vitro. Unlike Cas9 nucleases that cleaved both strands of the DNA substrate, Cas9 nickases composed of a guide RNA and a mutant form of Cas9 in which the catalytic aspartic acid residue was changed to an alanine (D10A Cas9) cleaved only one strand, resulting in a site-specific nick (Fig. S16c, d). Interestingly, however, some nickases (AS1, AS2, AS3, and S6 in Fig. S17a) induced indels at the target site in human cells, suggesting that nicks can be converted to DSBs in vivo, albeit inefficiently. Paired Cas9 nickases that make two adjacent nicks on opposing DNA strands generated indels at frequencies between 14% and 91%, comparable to the effect of paired nucleases (Fig. S17a). Repair of two nicks creating 5' overhangs led to indel formation at three genomic loci much more frequently than nicks creating 3' overhangs (Figure 17a and Figure 18). In addition, paired nickases enabled targeted genome editing by homology-directed repair more efficiently than single nickases (Figure 19).

[0104] We next measured the mutation frequencies of paired nickases and nucleases at off-target sites using deep sequencing. Cas9 nuclease complexed with three sgRNAs induced off-target mutations at six sites that differed from their corresponding on-target sites by one or two nucleotides, with frequencies ranging from 0.5% to 10% (Figure 17b). In contrast, paired Cas9 nickase did not produce indels above the detection limit of 0.1% at any of the six off-target sites. The S2 Off-1 site, which differs from its on-target site by one nucleotide at the first position in the PAM (i.e., N in NGG), can be considered another on-target site. As expected, Cas9 nuclease complexed with S2 sgRNA was similarly effective at this site and the on-target site. In sharp contrast, D10A Cas9 complexed with S2 and AS2 sgRNAs discriminated this site from the on-target site by a 270-fold difference. The paired nickase also discriminated the AS2 off-target sites (Off-1 and Off-9 in Figure 17b) from the on-target site by a 160-fold and 990-fold difference, respectively. EXAMPLES

[0105] Chromosomal DNA splicing induced by paired Cas9 nickases It has been reported that two simultaneous DSBs generated by artificial nucleases such as ZFNs and TALENs can promote large deletions of intervening chromosomal segments. We tested whether two SSBs caused by paired Cas9 nickases can also generate deletions in human cells. We detected the deletion events using PCR and found that seven paired nickases induced deletions of chromosomal segments up to 1.1 kbp as efficiently as paired Cas9 nucleases (Fig. 20a, b). The deletion events were confirmed by DNA sequencing of the PCR products (Fig. 20c). Interestingly, the sequence matching the sgRNA was completely retained in two of the seven deletion-specific PCR amplicons (underlined in Fig. 20c). In contrast, the Cas9 nuclease pair did not generate a sequence containing the complete target site. This finding suggests that the two distant nicks were not converted into two independent DSBs to promote the deletion of the intervening chromosomal segment. In addition, the melting temperature is so high that it is unlikely that two nicks more than 100 bp apart could give rise to a compound DSB with a large overhang under physiological conditions.

[0106] We propose that two separate nicks are repaired by strand displacement in a head-to-head orientation, leading to the formation of a central DSB, whose repair by NHEJ causes a small deletion (Fig. 20d). During this process, the two target sites remain intact, so the nickase again creates an SSB, triggering this cycle repeatedly until the target site is deleted. This mechanism explains why two offset nicks creating 5' overhangs, but not nicks creating 3' overhangs, efficiently induced indels at the three loci.

[0107] We then investigated whether Cas9 nuclease and nickase could induce undesired chromosomal translocations due to NHEJ repair of on-target and off-target DNA breaks (Fig. 21a). We were able to detect the translocations induced by Cas9 nuclease using PCR (Fig. 21b, c). Such PCR products were not amplified using genomic DNA isolated from cells transfected with plasmids encoding the AS2+S3 Cas9 nickase pair. This result is consistent with the fact that both AS2 and S3 nickases, unlike their corresponding nucleases, did not induce indels at off-target sites (Fig. 17b).

[0108] These results suggest that paired Cas9 nickases enable targeted mutagenesis and large deletions of chromosomal segments up to 1-kbp in human cells. Importantly, paired nickases did not cause indels at off-target sites where their corresponding nucleases induce mutations. Furthermore, unlike nucleases, paired nickases did not promote undesired translocations associated with off-target DNA cleavage. In principle, paired nickases double the specificity of Cas9-mediated mutagenesis, expanding the utility of RNA-guided enzymes in applications requiring precise genome editing, such as gene and cell therapy. One caveat with this approach is that two highly active sgRNAs are required to create an efficient nickase pair, limiting the sites that can be targeted. As shown in this and other studies, not all sgRNAs are equally active. If single clones, rather than populations of cells, are used for further studies or applications, the selection of guide RNAs that display unique sequences in the genome and the use of optimized guide RNAs will be sufficient to avoid off-target mutations associated with Cas9 nuclease.We propose that both Cas9 nuclease and paired nickases are powerful options to facilitate precise genome editing in cells and organisms. EXAMPLES

[0109] Genotyping using CRISPR / Cas-derived RNA-guided endonucleases Next, we reasoned that RGEN could be used in restriction fragment length polymorphism (RFLP) analysis in place of traditional restriction enzymes. Artificial nucleases, including RGEN, induce indels at target sites when the DSBs introduced by the nucleases are repaired by the error-prone non-homologous end joining (NHEJ) system. RGENs designed to recognize the target sequence will not be able to cut mutant sequences with indels, but will effectively cut wild-type target sequences.

[0110] 9-1. RGEN component crRNA and tracrRNA were prepared by in vitro transcription using the MEGAshortcript T7 kit (Ambion) according to the manufacturer's instructions. The transcribed RNA was separated on an 8% denaturing urea-PAGE gel. The gel slice containing the RNA was excised and transferred to elution buffer. The RNA was recovered in nuclease-free water, followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. The purified RNA was quantified by spectrometry. The template for crRNA was 5'-GAAATTAATACGACTCACTATAGGX 20 GTTTTAGAGCTATGCTGTTTTG-3' (SEQ ID NO: 76) (X 20 The template for tracrRNA was prepared by annealing the oligonucleotides shown as , and their complementary oligonucleotides ( , , ). The template for tracrRNA was prepared by annealing the forward and reverse oligonucleotides using Phusion polymerase (New England Biolabs). (5'-GAAATTAATACGACTCACTATAGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCG-3' (SEQ ID NO: 77) and The oligonucleotide was synthesized by extension of 5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATG-3' (SEQ ID NO: 78).

[0111] 9-2. Purification of recombinant Cas9 protein The Cas9 DNA construct used in our previous example, encoding Cas9 fused with a His6-tag at the C-terminus, was inserted into the pET-28a expression vector. The recombinant Cas9 protein was expressed in E. coli strain BL21(DE3) grown in LB medium for 4 hours at 25°C after induction with 1 mM IPTG. The cells were harvested and resuspended in a buffer containing 20 mM Tris PH 8.0, 500 mM NaCl, 5 mM imidazole, and 1 mM PMSF. The cells were frozen in liquid nitrogen, thawed at 4°C, and sonicated. After centrifugation, the Cas9 protein in the lysate was bound to Ni-NTA agarose resin (Qiagen), washed with a buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 20 mM imidazole, and eluted with a buffer containing 20 mM Tris pH 8.0, 500 mM NaCl, and 250 mM imidazole. Purified Cas9 protein was dialyzed against 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, and 10% glycerol and analyzed by SDS-PAGE.

[0112] 9-3. T7 endonuclease I assay The T7E1 assay was performed as follows: Briefly, PCR products amplified using genomic DNA were denatured at 95°C, reannealed at 16°C, and incubated with 5 units of T7 endonuclease I (New England BioLabs) for 20 min at 37°C. Reaction products were separated using 2-2.5% agarose gel electrophoresis.

[0113] 9-4. RGEN-RFLP assay PCR products (100–150 ng) were incubated in 10 μl of NEB buffer 3 (1×) with optimized concentrations (Table 10) of Cas9 protein, tracrRNA, and crRNA for 60 min at 37°C. After the cleavage reaction, RNase A (4 μg) was added and the reaction mixture was incubated at 37°C for 30 min to remove RNA. The reaction was stopped by 6× stop buffer containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were separated by 1–2.5% agarose gel electrophoresis and visualized by EtBr staining. [Table 10] [Table 11]

[0114] 9-5. Plasmid cleavage assay The restricted linearized plasmid (100 ng) was incubated with Cas9 protein (0.1 μg), tracrRNA (60 ng), and crRNA (25 ng) in 10 μl of NEB 3 buffer (1×) for 60 min at 37° C. The reaction was stopped with 6× stop solution containing 30% glycerol, 1.2% SDS, and 100 mM EDTA. Products were separated by 1% agarose gel electrophoresis and visualized by EtBr staining.

[0115] 9-6. RFLP strategy New RGENs with desired DNA specificity can be easily created by replacing the crRNA, and once the recombinant Cas9 protein is obtained, de novo purification of custom proteins is not required. Artificial nucleases containing RGENs induce small insertions or deletions (indels) at the target site when the DSB caused by the nuclease is repaired by error-prone non-homologous end joining (NHEJ). RGENs designed to recognize the target sequence efficiently cleave the wild-type sequence but cannot cleave the mutant sequence with indels (Figure 22).

[0116] We first tested whether RGEN could differentially cleave plasmids containing wild-type or modified C4BPB target sequences with 1- to 3-base indels at the cleavage site. None of the six plasmids with these indels were cleaved by the C4BPB-specific RGEN5, which is composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein (Figure 23). In contrast, plasmids with the complete target sequence were efficiently cleaved by this RGEN.

[0117] 9-7. Detection of mutations induced by the same RGEN using RGEN-mediated RFLP Next, to test the feasibility of RGEN-mediated RFLP for detection of the same RGEN-induced mutations, we used genetically modified K562 human cancer cell clones established with RGEN targeting the C4BPB gene (Table 12). [Table 12]

[0118] The C4BPB mutant clones used in this study have various mutations ranging from a 94 bp deletion to a 67 bp insertion (Figure 24a). Importantly, all mutations that occurred in the mutant clones resulted in the absence of the RGEN target site. Among the six C4BPB clones analyzed, four clones had both wild-type and mutant alleles (+ / -), and two clones had only mutant alleles (- / -).

[0119] PCR products spanning the RGEN target site amplified from wild-type K562 genomic DNA were completely digested by RGEN, which is composed of target-specific crRNA, tracrRNA, and recombinant Cas9 protein expressed and purified in E. coli (Figure 24b / lane 1). When C4BPB mutant clones were subjected to RFLP analysis with RGEN, the PCR amplicons of + / - clones carrying both wild-type and mutant alleles were partially digested, while the amplicons of - / - clones not carrying the wild-type allele were not digested at all, resulting in no cleavage products corresponding to the wild-type sequence (Figure 24b). Even a single base insertion at the target site prevented digestion of the amplified mutant allele by C4BPB RGEN (#12 and #28 clones), demonstrating the high specificity of RGEN-mediated RFLP. We simultaneously subjected the PCR amplicons to a mismatch-sensitive T7E1 assay (Figure 24b). Notably, the T7E1 assay was unable to distinguish - / - clones from + / - clones. To make matters worse, the T7E1 assay cannot distinguish homozygous mutant clones with the same mutant sequence from wild-type clones because annealing of the same mutant sequence forms a homoduplex. Therefore, RGEN-mediated RFLP has an important advantage over conventional mismatch-sensitive nuclease assays in analyzing mutant clones induced by artificial nucleases such as ZFN, TALEN, and RGEN.

[0120] 9-8. Quantitative assay for RGEN-RFLP analysis We also investigated whether RGEN-RFLP analysis is a quantitative method. Genomic DNA samples isolated from C4BPB null clones and wild-type cells were mixed in various ratios and used for PCR amplification. The PCR products were simultaneously subjected to RGEN genotyping and T7E1 assay (Figure 25b). As expected, DNA cleavage by RGEN was proportional to the ratio of wild-type to mutant. In contrast, the results of the T7E1 assay were poorly correlated with the mutation frequency estimated from the ratio and were inaccurate, especially at high mutant %, a situation in which complementary mutant sequences can hybridize with each other to form homoduplexes.

[0121] 9-9. Analysis of mutant mouse founders using RGEN-mediated RFLP genotyping We also applied RGEN-mediated RFLP genotyping (RGEN genotyping for short) to the analysis of mutant mouse founders established by injection of TALENs into mouse 1-cell embryos (Figure 26a). We designed and used RGENs that recognize the TALEN target site in the Pibf1 gene (Table 10). Genomic DNA was isolated from wild-type and mutant mice and subjected to RGEN genotyping after PCR amplification. RGEN genotyping successfully detected various mutations ranging from 1 to 27-bp deletions (Figure 26b). Unlike the T7E1 assay, RGEN genotyping allowed for the discriminatory detection of + / - and - / - founders.

[0122] 9-10. Detection of mutations induced in human cells by CCR5-specific ZFNs using RGEN In addition, we used RGEN to detect mutations induced in human cells by yet another type of artificial nuclease, CCR5-specific ZFN (Figure 27). These results indicate that RGEN can detect mutations induced by nucleases other than RGEN itself. In fact, we expect that RGEN can be designed to detect mutations induced by most, but not all, artificial nucleases. The only constraint in the design of the RGEN genotyping assay is the requirement for a GG or AG (CC or CT in the complementary strand) dinucleotide in the PAM sequence recognized by the Cas9 protein, which occurs on average once every 4 bp. Indels induced anywhere within the seed region of several bases in the crRNA and PAM nucleotides are expected to prevent RGEN-catalyzed DNA cleavage. In fact, we confirmed at least one RGEN site in most (98%) of the ZFN and TALEN sites.

[0123] 9-11. Detection of polymorphism or diversity using RGEN Next, we designed and tested a new RGEN targeting HLA-B, which encodes human leukocyte antigen B (also known as MHC class I protein), a highly polymorphic locus (Figure 28). HeLa cells were transfected with the RGEN plasmid and genomic DNA was simultaneously subjected to T7E1 assay and RGEN-RFLP analysis. T7E1 produced a false positive band due to sequence polymorphisms close to the target site (Figure 25c). However, as expected, the same RGEN used for gene disruption completely cleaved the PCR product from wild-type cells but partially cleaved the PCR product from RGEN-transfected cells, indicating the presence of RGEN-induced indels at the target site. This result indicates that RGEN-RFLP analysis has clear advantages over T7E1 assay, especially when it is unknown whether the target gene has polymorphism or diversity in the cells of interest.

[0124] 9-12. Detection of recurrent mutations and naturally occurring polymorphisms in cancer by RGEN-RFLP analysisRGEN-RFLP analysis has applications beyond genotyping artificial nuclease-induced mutations. We attempted to use RGEN genotyping to detect recurrent mutations and naturally occurring polymorphisms found in cancer. We selected the human colon cancer cell line HCT116, which has a gain-of-function 3-bp deletion in the oncogenic CTNNB1 gene that encodes β-catenin. PCR products amplified from HCT116 genomic DNA were partially cleaved by both wild-type-specific and mutation-specific RGENs, consistent with the heterozygous genotype in HCT116 cells (FIG. 29a). In clear contrast, PCR products amplified from DNA from HeLa cells carrying only the wild-type allele were completely digested by wild-type-specific RGENs and not cleaved at all by mutation-specific RGENs.

[0125] We also noted that HEK293 cells carry a 32-bp deletion (del32) in the CCR5 gene, which encodes a coreceptor essential for HIV infection, and homozygous del32 CCR5 carriers are immune to HIV infection. We designed one RGEN specific for the del32 allele and the other for the wild-type allele. As expected, the wild-type-specific RGEN completely cleaved the PCR products obtained from K562, SKBR3, or HeLa cells (used as wild-type controls), but partially cleaved the PCR products from HEK293 cells (Figure 30a), confirming the presence of the uncleavable del32 allele in HEK293 cells. However, unexpectedly, the del32-specific RGEN cleaved the PCR products from wild-type cells as efficiently as the PCR products from HEK293 cells. Interestingly, this RGEN had an off-target site with a single-base mismatch immediately downstream of the on-target site (Figure 30). These results suggest that RGEN can detect naturally occurring indels but cannot distinguish sequences with single nucleotide polymorphisms or point mutations due to their off-target effects.

[0126] To genotype oncogenic single nucleotide mutations using RGEN, we weakened RGEN activity by using a single-base mismatch guide RNA instead of a perfectly matched RNA. RGENs containing perfectly matched guide RNAs specific to the wild-type or mutant sequence cleaved both sequences (Figures 31a and 32a). In contrast, RGENs containing a single-base mismatch guide RNA distinguished between the two sequences, allowing genotyping of three recurrent oncogenic point mutations in the KRAS, PIK3CA, and IDH1 genes in human cancer cell lines (Figure 29b and Figures 33a, b). In addition, we were able to detect point mutations in the BRAF and NRAS genes using RGENs that recognize the NAG PAM sequence (Figures 33c, d). We believe that by using RGEN-RFLP, it is possible to genotype almost any, if not all, mutations or polymorphisms in human and other genomes.

[0127] The above data propose RGEN as a basis for using simple and reliable RFLP analysis for various sequence variations. Due to its high flexibility in reprogramming target sequences, RGEN can be used to detect various genetic variations (single base mutations, small insertions / deletions, structural mutations), such as disease-related recurrent mutations, genotypes related to patient drug responses, and mutations induced by artificial nucleases in cells. Herein, we have used RGEN genotyping to detect mutations induced by artificial nucleases in cells and animals. In principle, RGEN could be used to specifically detect and cleave naturally occurring polymorphisms and mutations.

[0128] Based on the above description, it should be understood by those skilled in the art that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention without departing from the technical idea or essential features of the present invention as defined in the following claims. In this regard, the above examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all modifications or variations that fall within the spirit and scope of the following claims or their equivalent concepts.

[0129] References 1. M. Jinek et al., Science 337, 816 (Aug 17, 2012). 2. H. Kim, E. Um, SR Cho, C. Jung, JS Kim, Nat Methods 8, 941 (Nov, 2011).3. HJ Kim, HJ Lee, H. Kim, SW Cho, JS Kim, Genome Res 19, 1279 (Jul, 2009). 4. EE Perez et al., Nat Biotechnol 26, 808 (Jul, 2008). 5. JC Miller et al., Nat Biotechnol 29, 143 (Feb, 2011). 6. C. Mussolino et al., Nucleic Acids Res 39, 9283 (Nov, 2011). 7. J. Cohen, Science 332, 784 (May 13, 2011). 8. V. Pattanayak, CL Ramirez, JK Joung, DR Liu, Nat Methods 8, 765 (Sep, 2011). 9. R. Gabriel et al., Nat Biotechnol 29, 816 (Sep, 2011). 10. E. Kim et al., Genome Res, (Apr 20, 2012). 11. H. J. Lee, J. Kweon, E. Kim, S. Kim, J. S. Kim, Genome Res 22, 539 (Mar, 2012). 12. H. J. Lee, E. Kim, J. S. Kim, Genome Res 20, 81 (Jan, 2010). 13. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotech advance online publication (2013) JPEG2025072369000017.jpg245137 JPEG2025072369000018.jpg244135 JPEG2025072369000019.jpg244145 JPEG2025072369000020.jpg245140 JPEG2025072369000021.jpg243136 JPEG2025072369000022.jpg245144 JPEG2025072369000023.jpg244145 JPEG2025072369000024.jpg245153 JPEG2025072369000025.jpg243143 JPEG2025072369000026.jpg244146 JPEG2025072369000027.jpg244142 JPEG2025072369000028.jpg245142 JPEG2025072369000029.jpg244137 JPEG2025072369000030.jpg244158 JPEG2025072369000031.jpg244148 JPEG2025072369000032.jpg245144 JPEG2025072369000033.jpg243146 JPEG2025072369000034.jpg244148 JPEG2025072369000035.jpg245139 JPEG2025072369000036.jpg244150 JPEG2025072369000037.jpg243149 JPEG2025072369000038.jpg245146 JPEG2025072369000039.jpg244145 JPEG2025072369000040.jpg243135 JPEG2025072369000041.jpg245143 JPEG2025072369000042.jpg244138 JPEG2025072369000043.jpg245134 JPEG2025072369000044.jpg244125 JPEG2025072369000045.jpg243142 JPEG2025072369000046.jpg244134 JPEG2025072369000047.jpg244138 JPEG2025072369000048.jpg244130 JPEG2025072369000049.jpg244132 JPEG2025072369000050.jpg244151

Claims

1. A method for producing a non-human mammal having engineered mammalian cells, comprising: providing at least one Cas9 / RNA complex, wherein said Cas9 / RNA complex comprises a Cas9 protein and a guide RNA having a CRISPR RNA (crRNA) and a transcription-activating crRNA (tracrRNA), said crRNA comprising i) a first portion that hybridizes to a portion of the tracrRNA and ii) a second portion that is complementary to a target DNA; introducing the Cas9 / RNA complex into a mammalian cell; allowing the Cas9 / RNA complex to modify a target DNA to provide an engineered mammalian cell comprising a nucleus comprising at least a partially modified target DNA; and administering the engineered mammalian cells to a non-human mammal; Including, The engineered mammalian cells are free of plasmid DNA. The method.

2. 2. The method of claim 1, wherein the engineered mammalian cells are derived from a non-human mammal prior to said engineering.

3. The method of claim 1 , wherein the engineered mammalian cell is a pluripotent cell.

4. 2. The method of claim 1, wherein the Cas9 / RNA complex is assembled in vitro.

5. 2. The method of claim 1, wherein the guide RNA is a single-stranded guide RNA (sgRNA) comprising a crRNA fused to a tracrRNA.

6. The sequence of the target DNA is i) a first strand having a 20 base pair region complementary to a second portion of the crRNA; and ii) a second strand with a trinucleotide protospacer adjacent motif (PAM) The method of claim 1 , comprising:

7. 2. The method of claim 1, wherein the Cas9 protein is a purified recombinant protein expressed by bacteria.

8. 2. The method of claim 1, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein.

9. 2. The method of claim 1, wherein the guide RNA is transcribed in vitro.

10. 2. The method of claim 1, wherein the guide RNA is chemically synthesized.

11. The method of claim 1 , wherein the target DNA is at least partially modified.

12. 2. The method of claim 1, wherein the target DNA has one or more double stranded breaks (DSBs).

13. 2. The method of claim 1, wherein the target DNA has one or more indels.

14. 2. The method of claim 1, wherein the target DNA is substantially free of off-target mutations.

15. 1. A method for producing a non-human mammal having a modified target endogenous nucleic acid, comprising: providing at least one Cas9 / RNA complex, wherein said Cas9 / RNA complex comprises a Cas9 protein and a single-stranded guide RNA (sgRNA) comprising a crRNA and a tracrRNA, and wherein the target endogenous nucleic acid comprises a portion of the guide RNA that is complementary to the crRNA; introducing the Cas9 / RNA complex into a mammalian cell; allowing the Cas9 / RNA complex to modify a target endogenous nucleic acid to provide a modified mammalian cell; and administering the modified mammalian cells to a non-human mammal; The method comprising:

16. The method of claim 15, wherein the mammalian cell is derived from the non-human mammal.

17. The method of claim 15, wherein the mammalian cell is a pluripotent cell.

18. 16. The method of claim 15, wherein the Cas9 / RNA complex is assembled in vitro.

19. 16. The method of claim 15, wherein the Cas9 protein is a purified recombinant protein expressed by bacteria.

20. 16. The method of claim 15, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein.

21. 16. The method of claim 15, wherein the sgRNA is transcribed in vitro.

22. The method of claim 15, wherein the sgRNA is chemically synthesized.

23. 16. The method of claim 15, wherein the target endogenous nucleic acid in the modified mammalian cell is at least partially modified.

24. 16. The method of claim 15, wherein the target endogenous nucleic acid in the modified mammalian cell has one or more double-stranded breaks (DSBs).

25. 16. The method of claim 15, wherein the target endogenous nucleic acid in the modified mammalian cell has one or more indels.

26. 16. The method of claim 15, wherein the target endogenous nucleic acid in the modified mammalian cell is substantially free of off-target mutations.

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