A composition for cleaving target DNA, comprising a guide RNA specific to the target DNA and a CAS protein-coding nucleic acid or CAS protein, and its use.

Abstract

To provide a method for targeted genome editing in eukaryotic cells or organisms.SOLUTION: Provided is a method for inducing modification of a target nucleic acid sequence in a plant cell, the method comprising: obtaining an artificial and / or non-naturally occurring type II CRISPR / Cas9 complex, the CRISPR / Cas9 complex being a combination of protein and RNA, the protein being a Cas9 protein, and the RNA being a guide RNA; and introducing the CRISPR / Cas9 complex into the plant cell by transfection using a transfection buffer comprising polyethylene glycol (PEG), the guide RNA, which comprises a crRNA portion and a tracrRNA portion, being transcribed in vitro or chemically synthesized, and the target nucleic acid sequence, which is endogenous DNA, comprising a portion complementary to the crRNA portion of the guide RNA.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

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

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a locus containing numerous short serial repeats found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR functions as the immune system in prokaryotes, where it confers resistance to exogenous genetic factors such as plasmids and phages. The CRISPR system results in a type of adaptive immunity. Short segments of foreign DNA, called spacers, are incorporated between CRISPR repeats in the genome and function as a memory of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic factors 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 it complexes with two RNAs called CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), thereby defending host cells by cleaving invading phages or foreign genetic elements in plasmids. crRNA is transcribed from CRISPR elements in the host genome that have been previously captured from such invaders. Recently, Jinek et al. (1) demonstrated that single-stranded chimeric RNA produced by the fusion of essential regions of crRNA and tracrRNA can replace the two RNAs in the Cas9 / RNA complex and form a functional endonuclease.

[0004] The site specificity in nucleotide-binding CRISPR-Cas proteins is governed by RNA molecules, rather than DNA-binding proteins, which can be more difficult to design and synthesize. Therefore, the CRISPR / Cas system offers advantages over zinc finger and transcription activator-like effector DNA-binding proteins. However, until now, genome editing methods using RNA-induced endonucleases (RGENs) based on the CRISPR / Cas system had not been developed. On the other hand, restriction fragment length polymorphism (RFLP), still widely used in molecular biology and genetics, is one of the oldest, simplest, and cheapest genotyping methods, but it is often limited by the lack of suitable sites recognized by restriction endonucleases.

[0005] Engineered nuclease-induced mutations are detected by various methods, including mismatch-sensitive T7 endonuclease I (T7E1) assays or Surveyor nuclease assays, RFLP, capillary electrophoresis of fluorescent PCR products, dideoxy sequencing, and deep sequencing. While T7E1 and Surveyor assays are widely used, they are cumbersome. Furthermore, these enzymes tend to underestimate mutation frequencies because mutant sequences can form homoduplexes with each other, making it impossible to distinguish between homozygous biallele mutant clones and wild-type cells. RFLP is preferred because it does not have these limitations. In fact, RFLP was one of the first methods for detecting engineered nuclease-mediated mutations in cells and animals. Unfortunately, however, RFLP is limited by the availability of appropriate restriction enzyme recognition sites. There may be no restriction enzyme recognition sites available at the target site of interest. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Until now, genome editing and genotyping methods using RNA-induced endonucleases (RGENs) based on the CRISPR / Cas system had not been developed. Under these circumstances, the inventors have made considerable efforts to develop genome editing methods based on the CRISPR / Cas system and have finally established a programmable RNA-induced endonuclease that targets and cleaves DNA in eukaryotic cells and eukaryotes. In addition, the inventors have made considerable efforts to develop a novel method for using RNA-induced endonuclease (RGEN) in RFLP analysis. Using RGEN, the inventors performed genotyping of recurrent mutations found in cancer, as well as mutations induced in cells and organisms by artificial nucleases, including RGEN itself, thereby completing the present invention. [Means for solving the problem]

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

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

[0009] A further object of the present invention is to provide a method for producing eukaryotic cells or eukaryotes having Cas protein and guide RNA, comprising the step of simultaneously or sequentially transfecting eukaryotic cells or eukaryotes with Cas protein-coding nucleic acid or Cas protein and guide RNA or DNA encoding guide RNA. A further object of the present invention is to provide a eukaryotic cell or eukaryote containing a guide RNA or DNA encoding a guide RNA that is specific to a target DNA, and a Cas protein-coding nucleic acid or Cas protein.

[0010] A further object of the present invention is to provide a method for cleaving target DNA in a eukaryotic cell or eukaryote, comprising the step of transfecting a eukaryotic cell or eukaryote containing target DNA with a composition containing a guide RNA or DNA encoding a guide RNA specific to target DNA, and a Cas protein-coding nucleic acid or Cas protein. A further object of the present invention is to provide a method for inducing targeted mutagenesis in eukaryotic cells or eukaryotes, comprising the step of treating eukaryotic cells or eukaryotes with a composition containing a guide RNA or DNA encoding a guide RNA specific to target DNA, and a Cas protein-coding nucleic acid or Cas protein.

[0011] A further object of the present invention is to provide an embryo, a genetically modified animal, or a genetically modified plant having a genome edited with a composition containing a guide RNA or DNA encoding a guide RNA specific to a target DNA, and a Cas protein-coding nucleic acid or Cas protein. A further object of the present invention is to provide a method for producing a genome-modified animal, comprising the steps of introducing a composition containing a guide RNA or DNA encoding a guide RNA specific to a target DNA, and a Cas protein-coding nucleic acid or Cas protein into an animal embryo; and transferring the embryo into the fallopian tube of a pseudopregnant surrogate mother to produce a genome-modified animal.

[0012] A further object of the present invention is to provide a composition for genotyping mutations or polymorphisms in isolated biological samples, comprising a guide RNA and Cas protein specific to a target DNA sequence. A further object of the present invention is to provide a method for genotyping mutations or polymorphisms induced in cells by artificial nucleases or spontaneous mutations or polymorphisms, wherein the RGEN comprises a target DNA-specific guide RNA and a Cas protein.

[0013] Yet another object of the present invention is to provide a kit containing an RNA-guided endonuclease (RGEN) for genotyping mutations or spontaneous mutations or polymorphisms induced intracellularly by artificial nuclease, wherein THE RGEN contains a guide RNA specific to the 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, which contains a guide RNA specific to the target DNA or 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 a eukaryotic cell or organism, which contains a guide RNA specific to the target DNA or 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, which contains a guide RNA specific to the target DNA or 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 a eukaryotic cell or organism, which contains a guide RNA specific to the target DNA or 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 method for producing a eukaryotic cell or organism having a Cas protein and a guide RNA, which includes the step of co-transfecting or sequentially transfecting a eukaryotic cell or organism with a Cas protein-encoding nucleic acid or a Cas protein, and a guide RNA or DNA encoding the guide RNA.

[0016] Yet another object of the present invention is to provide a eukaryotic cell or eukaryote containing 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. Yet another object of the present invention is to provide a method for cleaving a target DNA in a eukaryotic cell or eukaryote, which comprises 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 eukaryote having the target DNA.

[0017] Yet another object of the present invention is to provide a method for inducing targeted mutagenesis in a eukaryotic cell or eukaryote, which comprises the step of treating the eukaryotic cell or eukaryote 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. Yet another object of the present invention is 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 the guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein.

[0018] Yet another object of the present invention is to provide a method for producing a genome-modified animal, which comprises the step of introducing a composition containing 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, into an embryo of an animal; and the step of transferring the embryo into the oviduct of a pseudopregnant surrogate mother to produce a genome-modified animal. Yet another object of the present invention is to provide a composition for genotyping a mutation or polymorphism in an isolated biological sample, which contains a guide RNA specific for a target DNA sequence and a Cas protein.

[0019] A further object of the present invention is to provide 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. A further object of the present invention is to provide a kit for genotyping mutations or polymorphisms in an isolated biological sample, comprising a composition, specifically an RNA-induced endonuclease (RGEN), wherein the RGEN comprises a target DNA-specific guide RNA and a Cas protein.

[0020] A further 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-induced endonuclease (RGEN), wherein the RGEN comprises a target DNA-specific guide RNA and a Cas protein. [Effects of the Invention]

[0021] The present composition, a kit containing the present composition, and a method for inducing targeted mutagenesis, all containing a target DNA-specific guide RNA and Cas protein-coding nucleic acid or Cas protein, for cleaving target DNA or inducing targeted mutagenesis in eukaryotic cells or eukaryotes, provide a novel and convenient genome editing tool. In addition, since custom RGEN can be designed to target any DNA sequence, virtually all single nucleotide polymorphisms or small insertions / deletions (indels) can be analyzed by RGEN-mediated RFLP, and therefore the compositions and methods of the present invention can be used for the detection and cleavage of spontaneous polymorphisms and mutations. [Brief explanation of the drawing]

[0022] [Figure 1a]Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (a) Schematic diagram of target DNA and chimeric RNA sequences. Red triangles indicate cleavage sites. PAM sequences recognized by Cas9 are shown in bold. Sequences in guide RNAs derived from crRNA and tracrRNA are shown in boxes and underlines, respectively. [Figure 1b] Figure 1 shows Cas9-catalyzed cleavage of plasmid DNA in vitro. (b) In vitro cleavage of plasmid DNA by Cas9. A complete (intact) circular plasmid or ApaLI digested plasmid was incubated with Cas9 and guide RNA. [Figure 2a] Figure 2 shows Cas9-induced mutagenesis at episomal target sites. (a) Schematic diagram of a cell-based assay using an RFP-GFP reporter. GFP is not expressed by this reporter because the GFP sequence is frameshifted and fused with the RFP sequence. The RFP-GFP fusion protein is expressed only when the target site between the two sequences is cleaved by a site-specific nuclease. [Figure 2b] Figure 2 shows Cas9-induced mutagenesis at episomal target sites. (b) Flow cytometry of Cas9-transfected cells. The percentage of cells expressing the 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. Arrows indicate the predicted locations of DNA bands cleaved by T7E1. Mutation frequency (Indels (%)) was calculated by measuring band intensity. (Bottom) DNA sequences of wild-type (WT) and mutant clones of CCR5 and C4BPB. Regions of target sequences complementary to the guide RNA are enclosed in a box (boc). PAM sequences are shown in bold. Triangles indicate cleavage sites. Bases corresponding to microhomology are underlined. The right-hand column shows the number of inserted or deleted bases. [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 locations of DNA bands cleaved by T7E1. Mutation frequency (Indels (%)) was calculated by measuring band intensity. (Bottom) DNA sequences of wild-type (WT) and mutant clones of CCR5 and C4BPB. Regions of target sequences complementary to the guide RNA are enclosed in a box (boc). PAM sequences are shown in bold. Triangles indicate cleavage sites. Bases corresponding to microhomology are underlined. The right-hand column shows the number of inserted or deleted bases. [Figure 4a] Figure 4 shows that no RGEN-induced off-target mutations were detected. (a) On-target and potential off-target sequences. The human genome was searched using a computer for potential off-target sites. Four sites were identified, each with a 3-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) The T7E1 assay was used to examine whether these sites mutated in cells transfected with the Cas9 / RNA complex. No mutations were detected at these sites. N / A (not applicable), intergenetic site. [Figure 4c] Figure 4 shows that no RGEN-induced off-target mutations were detected. (c) Cas9 did not induce off-target related chromosomal deletions. CCR5-specific RGEN and ZFN were expressed in human cells. PCR was used to detect the induction of 15-kb chromosomal deletions in these cells. [Figure 5a] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (a) Schematic diagram representing the sgRNA specific to exon 2 of the mouse Foxn1 gene. The PAM of exon 2 is shown in red, and the sequence in the sgRNA complementary to exon 2 is underlined. Triangles indicate cleavage sites. [Figure 5b] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (b) A representative T7E1 assay showing the 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)b DNA sequences of mutant alleles observed in the three Foxn1 mutant founders identified. The number of generations is shown in parentheses. [Figure 5d] Figure 5 shows RGEN-induced Foxn1 gene targeting in mice. (d) PCR genotyping of F1 offspring resulting from crossing Foxn1 founder #108 with wild-type FVB / NTac. Segregation of the mutant allele found in Foxn1 founder #108 is observed in the offspring. [Figure 6] Figure 6 shows Foxn1 gene targeting in mouse embryos by intracytoplasmic injection of Cas9 mRNA and Foxn1-sgRNA. (a) Representative results of the T7E1 assay measuring mutation rates after injection of the highest dose. Arrows indicate bands cleaved by T7E1. (b) Summary of T7E1 assay results. Shows 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 the wild-type allele are enclosed in a box. [Figure 7a] Figure 7 shows Foxn1 gene targeting in mouse embryos using a recombinant Cas9 protein:Foxn1-sgRNA complex. (a) and (b) are representative T7E1 assay results and their summaries. Embryos were cultured in vitro after pronuclear injection (a) or intracytoplasmic injection (b). Red numbers indicate T7E1-positive mutant founder mice. [Figure 7b]Figure 7 shows Foxn1 gene targeting in mouse embryos using a recombinant Cas9 protein:Foxn1-sgRNA complex. (a) and (b) are representative T7E1 assay results and their summaries. Embryos were cultured in vitro after pronuclear injection (a) or intracytoplasmic injection (b). Red numbers indicate T7E1-positive mutant founder mice. [Figure 7c] Figure 7 shows Foxn1 gene targeting in mouse embryos using the recombinant Cas9 protein:Foxn1-sgRNA complex. (c) DNA sequences of Foxn1 mutant alleles identified from embryos obtained by pronuclear injection of the highest dose of the recombinant Cas9 protein:Foxn1-sgRNA complex and cultured in vitro. Target sequences of the wild-type allele are shown in a box. [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 offspring. [Figure 9] Figure 9 shows the genotypes of embryos produced by mating Prkdc mutant founders. Prkdc mutant founders ♂25 and ♀15 ​​were mated, and E13.5 embryos were extracted. (a) fPCR analysis of wild type, founder ♂25, and founder ♀15. It should be noted that, due to the technical limitations of fPCR analysis, these results show slight differences from the exact sequences of the mutant alleles; for example, sequence analysis identified Δ269 / Δ61 / WT and Δ5+1 / +7 / +12 / WT in founder ♂25 and ♀15, respectively. (b) Genotypes of the resulting embryos. [Figure 10a] Figure 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10b] Figure 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10c] Figure 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10d] Figure 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 10e] Figure 10 shows Cas9 protein / sgRNA complex-induced targeted mutations. [Figure 11] Figure 11 shows recombinant Cas9 protein-induced mutations in Arabidopsis protoplasts. [Figure 12] Figure 12 shows recombinant Cas9 protein-induced mutation sequences in the Arabidopsis BRI1 gene. [Figure 13] Figure 13 shows the T7E1 assay, which demonstrates disruption of the endogenous CCR5 gene in 293 cells by treatment with Cas9-mal-9R4L and the sgRNA / C9R4LC complex. [Figure 14a] Figure 14(a, b) shows the mutation frequencies at on-target and off-target sites of RGEN as reported by Fu et al. (2013). T7E1 assay analysis of genomic DNA from K562 cells (1 × 10⁶ cells) sequentially transfected with (R) 20 μg of a Cas9-encoding plasmid and 60 μg and 120 μg of in vitro transcribed GX19 crRNA and tracrRNA, respectively, or (D) 1 μg of a Cas9-encoding plasmid and 1 μg of a GX19 sgRNA expression plasmid (2 × 10⁵ cells). [Figure 14b] Figure 14(a, b) shows the mutation frequencies at on-target and off-target sites of RGEN as reported by Fu et al. (2013). T7E1 assay analysis of genomic DNA from K562 cells (1 × 10⁶ cells) sequentially transfected with (R) 20 μg of a Cas9-encoding plasmid and 60 μg and 120 μg of in vitro transcribed GX19 crRNA and tracrRNA, respectively, or (D) 1 μg of a Cas9-encoding plasmid and 1 μg of a 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, as reported by Fu et al. (2013), was measured at on-target and off-target sites using the T7E1 assay. Plasmids encoding Cas9 and plasmids encoding GX19 sgRNA or GGX20 sgRNA were co-transfected into K562 cells. Off-target sites (OT1-3, etc.) were 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, as reported by Fu et al. (2013), was measured at on-target and off-target sites using the T7E1 assay. Plasmids encoding Cas9 and plasmids encoding GX19 sgRNA or GGX20 sgRNA were co-transfected into K562 cells. Off-target sites (OT1-3, etc.) were labeled as described in Fu et al. (2013). [Figure 16a] Figure 16 shows in vitro DNA cleavage by Cas9 nickases. (a) Schematic diagrams of Cas9 nuclease and paired Cas9 nickases. PAM sequences and cleavage sites are indicated by boxes. [Figure 16b] Figure 16 shows in vitro DNA cleavage by Cas9 nickase. (b) Target sites within the human AAVS1 gene 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 diagram of the DNA cleavage reaction. The FAM dye (indicated by the box) was bound to both 5' ends of the DNA substrate. [Figure 16d] Figure 16 shows in vitro DNA cleavage by Cas9 nickas. (d) DSBs and SSBs analyzed by fluorescent capillary electrophoresis. Fluorescently labeled DNA substrates were incubated with Cas9 nuclease or nickas before electrophoresis. [Figure 17a]Figure 17 shows a comparison of the behavior of Cas9 nuclease and nickase. (a) On-target mutation frequencies associated with Cas9 nuclease (WT), nickase (D10A), and paired nickase. Paired nickase that will produce a 5' or 3' overhang is 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 nickase. A total of seven potential off-target sites for three sgRNAs were analyzed. [Figure 18a] Figure 18 shows paired Cas9 nickase 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 nicks tested at other endogenous human loci. (b) Genome editing activity at each target site was detected by the T7E1 assay. Repair of two nicks that would result in a 5' overhang caused indel formation at a much higher frequency than those that would result in a 3' overhang. [Figure 18c] Figure 18 shows paired Cas9 nickase tested at other endogenous human loci. (c) sgRNA target sites at human CCR5 and BRCA2 loci. PAM sequences are shown in red. [Figure 18d] Figure 18 shows paired Cas9 nicks tested at other endogenous human loci. (d) Genome editing activity at each target site was detected by the T7E1 assay. Repair of two nicks that would result in a 5' overhang caused indel formation at a much higher frequency than repair of those that would result in a 3' overhang. [Figure 19]Figure 19 shows that paired Cas9 nickase mediates homologous recombination. (a) Method for detecting homologous recombination. The donor DNA contained an XbaI restriction enzyme site between two homology arms, and the endogenous target site lacked this site. Homologous recombination sequences were detected using a PCR assay. Genomic DNA-specific primers were used to prevent amplification of contaminating donor DNA. (b) Efficiency of homologous recombination. Only the amplicons of the region where homologous recombination occurred were digested by XbaI; the efficiency of this method was measured using the intensity of the cleavage band. [Figure 20a] Figure 20 shows DNA splicing induced by paired Cas9 nickase. (a) Target sites of paired nickase at the human AAVS1 locus. Distances between the AS2 site and each of the other sites are shown. Arrows indicate PCR primers. [Figure 20b] Figure 20 shows DNA splicing induced by paired Cas9 nickase. (b) Genomic deletions detected using PCR. Asterisks indicate deletion-specific PCR products. [Figure 20c] Figure 20 shows DNA splicing induced by paired Cas9 nickase. (c) DNA sequences of deletion-specific PCR products obtained using AS2 and L1 sgRNA. Target site PAM sequences are enclosed in boxes, and sgRNA-corresponding sequences are shown in capital letters. Complete sgRNA-corresponding sequences are underlined. [Figure 20d] Figure 20 shows DNA splicing induced by paired Cas9 nickase. (d) Schematic model of paired Cas9 nickase-mediated chromosomal deletion. The newly synthesized DNA strand is shown in a box. [Figure 21a] Figure 21 shows that paired Cas9 nickase does not induce translocation. (a) Schematic diagram of chromosomal translocation between on-target and off-target sites. [Figure 21b] Figure 21 shows that paired Cas9 nickase does not induce translocation. (b) PCR amplification for detecting chromosomal translocation. [Figure 21c]Figure 21 shows that paired Cas9 nickase does not cause translocation. (c) Translocation is caused by Cas9 nuclease, but not by nickase pair. [Figure 22a] Figure 22 shows conceptual diagrams of the T7E1 assay and RFLP assay. (a) Comparison of assay cleavage reactions in four possible scenarios after artificial nuclease treatment in diploid cells: (A) wild type, (B) single allele mutation, (C) different biallele mutations (heterozygous), and (D) identical biallele mutations (homozygous). Black lines represent PCR products derived from each allele; dashed and dotted boxes indicate insertion / deletion mutations caused by NHEJ. [Figure 22b] Figure 22 shows conceptual diagrams of the T7E1 assay and RFLP assay. (b) Expected results of T7E1 digestion and RGEN digestion separated by electrophoresis. [Figure 23] Figure 23 shows an in vitro cleavage assay of a linearized plasmid containing an indel-containing C4BPB target site. The DNA sequences of individual plasmid substrates are shown (top panel). PAM sequences are underlined. Inserted bases are enclosed in boxes. Arrows (bottom panel) indicate the expected positions of DNA bands after electrophoresis following cleavage by wild-type-specific RGEN. [Figure 24] Figure 24 shows genotyping of cell-induced mutations by artificial nucleases using RGEN-mediated RFLP. (a) Genotype of C4BPB mutant K562 cell clone. (b) Comparison with RGEN-mediated RFLP analysis of mismatch-sensitive T7E1 assay. Black arrows indicate cleavage products by T7E1 enzyme or RGEN treatment. [Figure 25a] Figure 25 shows genotyping of RGEN-induced mutations using the RGEN-RFLP method. (a) Analysis of C4BPB disruption clones using RGEN-RFLP and T7E1 assays. Arrows indicate the expected locations of DNA bands cleaved by RGEN or T7E1. [Figure 25b]Figure 25 shows the 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 amplified by PCR. [Figure 25c] Figure 25 shows the 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 mouse. [Figure 26b] Figure 26 shows the genotyping of organism-induced mutations by artificial nucleases using RGEN-mediated RFLP. (b) Comparison with RGEN-mediated RFLP analysis of the mismatch-sensitive T7E1 assay. Black arrows indicate cleavage products by treatment with the T7E1 enzyme or RGEN. [Figure 27] Figure 27 shows RGEN-mediated genotyping of ZFN-induced mutations. ZFN target sites are indicated by boxes. Black arrows indicate DNA bands cleaved by T7E1. [Figure 28] Figure 28 shows polymorphic regions within the human HLA-B gene. The sequences surrounding the RGEN target site are from PCR amplicons from HeLa cells. The locations of the polymorphisms are indicated by boxes. The RGEN target site and PAM sequences are indicated by dashed and thick line boxes, respectively. Primer sequences are underlined. [Figure 29]Figure 29 shows the genotyping of oncogenic mutations using RGEN-RFLP analysis. (a) A repeat mutation (c.133-135 deletion of TCT) in the human CTNNB1 gene of HCT116 cells was detected by RGEN. HeLa cells were used as a negative control. (b) Genotyping of a KRAS substitution mutation (c.34 G>A) in the A549 cancer cell line using RGEN with mismatch guide RNA. Mismatch nucleotides are indicated by boxes. 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. Arrows indicate 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 the wild-type and delta32 CCR5 alleles. Both on-target and off-target sites of RGEN used in RFLP analysis are underlined. Single nucleotide mismatches between two sites are enclosed in boxes. PAM sequences are underlined. [Figure 30c] Figure 30 shows the genotyping of the CCR5 delta32 allele in HEK293T cells using RGEN-RFLP analysis. (c) In vitro cleavage of plasmids containing the WT or del32 CCR5 allele using wild-type specific RGEN. [Figure 30d]Figure 30 shows the 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 mutations (c.34 G>A). (a) RGEN-RFLP analysis of KRAS mutations (c.34 G>A) in cancer cell lines. PCR products from HeLa cells (used as wild-type control) or A549 cells (homozygous for point mutations) were digested by RGEN using fully matched crRNA specific to the wild-type or mutant sequence. The KRAS genotype in these cells was confirmed by Sanger sequencing. [Figure 31b] Figure 31 shows the genotyping of a KRAS point mutation (c.34 G>A). (b) Plasmids containing either wild-type or mutant KRAS sequences were digested by RGEN using perfectly matched crRNA or weakened single-nucleotide mismatch crRNA. Weakened crRNAs selected for genotyping are indicated in a box at the top of the gel. [Figure 32a] Figure 32 shows the genotyping of the PIK3CA point mutation (c.3140 A>G). (a) RGEN-RFLP analysis of the 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 using fully matched crRNA specific to 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 the wild-type or mutant PIK3CA sequence were digested by RGEN using perfectly matched crRNA or weakened single-nucleotide mismatch crRNA. Weakened crRNAs selected for genotyping are indicated in a box at the top of the gel. [Figure 33a] Figure 33 shows genotyping of repeating point mutations in cancer cell lines. (a) RGEN-RFLP assay of repeating 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 indicated by boxes. Black arrows indicate DNA bands cleaved by RGEN. [Figure 33b] Figure 33 shows genotyping of repeating point mutations in cancer cell lines. (b) RGEN-RFLP assay of repeating 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 indicated by boxes. Black arrows indicate DNA bands cleaved by RGEN. [Figure 33c] Figure 33 shows genotyping of repeating point mutations in cancer cell lines. (c) RGEN-RFLP assay of repeating 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 indicated by boxes. Black arrows indicate DNA bands cleaved by RGEN. [Figure 33d] Figure 33 shows genotyping of repeating point mutations in cancer cell lines. (d) RGEN-RFLP assay of repeating 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 indicated by boxes. Black arrows indicate DNA bands cleaved by RGEN. [Modes for carrying out the invention]

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

[0024] ZFNs and TALENs enable targeted mutagenesis in mammalian cells, model organisms, plants, and livestock, but the mutation frequencies produced by individual nucleases vary considerably. Furthermore, some ZFNs and TALENs do not exhibit genome editing activity. DNA methylation can limit the binding of these artificial nucleases to target sites. In addition, creating customized nucleases is technically difficult and time-consuming. The inventors have overcome the shortcomings of ZFNs and TALENs by developing a novel RNA-induced endonuclease composition based on Cas proteins.

[0025] Prior to this invention, the endonuclease activity of Cas proteins was known. However, due to the complexity of eukaryotic genomes, it was unknown whether the endonuclease activity of Cas proteins functioned in eukaryotic cells. Furthermore, until now, no composition for cleaving target DNA in eukaryotic cells or eukaryotes had been developed that contained Cas proteins or Cas protein-coding nucleic acids and guide RNA specific to target DNA.

[0026] Compared to ZFNs and TALENs, this RGEN composition based on Cas proteins can be more easily customized, as new genome editing nucleases can be created simply by replacing the synthetic guide RNA component. The subcloning step is not involved in the creation of customized RNA-inducible endonucleases. Furthermore, the relatively small size of the Cas gene (e.g., 4.2 kbp for Cas9) compared to a pair of TALEN genes (~6 kbp) offers advantages to this RNA-inducible endonuclease composition in several applications, such as virus-mediated gene delivery. Moreover, this RNA-inducible endonuclease has no off-target effects, thus not causing undesirable mutations, deletions, inversions, and duplications. These properties make this RNA-inducible endonuclease composition a scalable, versatile, and convenient tool for genome engineering in eukaryotic cells and eukaryotes. In addition, RGEN can be designed to target any DNA sequence, and virtually all single nucleotide polymorphisms or small insertions / deletions (indels) can be analyzed by RGEN-mediated RFLP. The specificity of RGEN is determined by the Cas9 protein, which recognizes RNA components and protospacer adjacent motifs (PAMs) that hybridize with target DNA sequences up to 20 base pairs (bp) in length. RGEN can be easily reprogrammed by replacing the RNA component. Therefore, RGEN provides a foundation for using simple and reliable RFLP analysis for a wide range of sequence mutations.

[0027] The target DNA may 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 that, when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA), forms an active endonuclease or nicasse. Information on the Cas gene and protein is freely available from the GenBank at the National Center for Biotechnology Information (NCBI).

[0028] CRISPR-associated (cas) genes encoding 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. Three types of CRISPR-Cas systems exist. Of these, the type ICRISPR / Cas system, which includes the Cas9 protein as well as 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 can bind to a protein transduction domain. This protein transduction domain may be, but is not limited to, polyarginine or a TAT protein derived from HIV. This composition may contain a Cas component in the form of a protein or in the form of a nucleic acid encoding a Cas protein. In this invention, a Cas protein can be any Cas protein, as long as it has endonuclease activity or nickase activity when it forms a complex with a guide RNA. Preferably, the Cas protein is the Cas9 protein or a variant thereof. A mutant of the Cas9 protein can be a mutation in which the catalytic aspartic acid residue within Cas9 is replaced with any other amino acid. Preferably, the other amino acid may be, but is not limited to, alanine.

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

[0031] The term "recombinant," when used, for example, in relation to cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins, or by alteration of native nucleic acids or proteins, or that the cells originate from such modified cells. For example, recombinant Cas proteins can be produced by reconstructing the Cas protein coding sequence using a human codon table.

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

[0033] As used herein, the term "guide RNA" refers to RNA that is specific to target DNA and can form a complex with the Cas protein to bring the Cas protein to the target DNA. In this invention, the guide RNA may consist of two RNAs, namely CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or it may be a single-stranded RNA (sgRNA) produced by the fusion of essential parts of crRNA and tracrRNA. Guide RNA can be a dual RNA containing both crRNA and tracrRNA. Any guide RNA can be used in the present invention as long as the guide RNA contains essential portions of crRNA and tracrRNA, as well as portions complementary to the target.

[0034] crRNA can hybridize to target DNA. RGENs consist of a Cas protein and dual RNA (an immutable tracrRNA and a target-specific crRNA), or a Cas protein and sgRNA (a fusion of an immutable tracrRNA and essential parts of a target-specific crRNA), and can be easily reprogrammed by crRNA replacement. The guide RNA further includes one or more additional nucleotides at the 5' end of the crRNA, whether it is a single-stranded guide RNA or a dual-RNA. Preferably, the guide RNA further includes two additional guanine nucleotides at the 5' end of the crRNA, whether single-stranded or dual-stranded.

[0035] Guide RNA can be introduced into a cell or organism in the form of RNA or DNA encoding the guide RNA. Guide RNA may be isolated RNA, RNA incorporated within a viral vector, or encoded by a vector. Preferably, the vector may 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 containing the sequence encoding the guide RNA. For example, the guide RNA can be introduced into a cell or organism by transfecting it with isolated guide RNA or plasmid DNA containing the sequences encoding the guide RNA and promoter. Alternatively, guide RNA can be introduced into cells or organisms using virus-mediated gene delivery.

[0036] When guide RNA is transfected into cells or organisms in the form of isolated RNA, the guide RNA may be prepared by in vitro transcription using any in vitro transcription system known in the art. Preferably, the guide RNA is transferred into cells in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA. Where used herein, the term “isolated RNA” may be replaced with “naked RNA.” This saves cost and time because it does not require a cloning step. However, the use of plasmid DNA or virus-mediated gene delivery for the transfection of guide RNA is not ruled out.

[0037] This RGEN composition, containing Cas protein or Cas protein-coding nucleic acid and guide RNA, can specifically cleave target DNA due to the specificity of the guide RNA to the target and the endonuclease activity or nickasase activity of the Cas protein. As used herein, the term "cleavage" refers to the cleavage of the covalent backbone of a nucleotide molecule. In this invention, the guide RNA can be prepared to be specific to any target to be cleaved. Therefore, 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” may refer to a pair of guide RNA and Cas protein. The pair comprises two guide RNAs. The guide RNA and Cas protein function as a pair, producing two nicks on different DNA strands. The two nicks may, but are not limited to, be at least 100 bp apart. In the examples, the inventors confirmed that paired Cas nickase enables targeted mutagenesis and large deletions of chromosomal segments up to 1-kbp in human cells. Importantly, paired nickase did not induce indels at off-target sites where their corresponding nucleases induce mutations. Furthermore, unlike nucleases, paired nickase did not promote undesirable translocations associated with off-target DNA cleavage. In principle, paired nickase doubles the specificity of Cas9-mediated mutagenesis and expands the utility of RNA-inducible enzymes in applications requiring precise genome editing, such as gene therapy and cell therapy.

[0039] In this invention, the composition can be used for genotyping of eukaryotic cells or the genome of eukaryotes in vitro. In one particular embodiment, the guide RNA may contain the nucleotide sequence of Sequence ID No. 1, but the portion of its nucleotide positions 3-22 is a target-specific region, and therefore the sequence of this portion may change depending on the target. As used herein, eukaryotic cells or eukaryotes may be, but are not limited to, yeast, fungi, protists, plants, higher plants, and insect or amphibian cells, as are commonly used in the art, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, for example, cultured cells (in vitro), transplanted cells and primary cultured cells (in vitro and ex vivo), and in vivo cells, as well as mammalian cells such as human cells.

[0040] In one specific embodiment, the Cas9 protein / single-strand guide RNA was found to be capable of inducing site-specific DNA double-strand breaks in vitro and in mammalian cells, and its spontaneous repair was found to frequently induce targeted genomic mutations. Furthermore, it was found that gene knockout mice can be induced by injecting the Cas9 protein / guide RNA complex or Cas9 mRNA / guide RNA into one-cell stage embryos, and that germline-transmittable mutations can be induced by the Cas9 / guide RNA system. Inducing targeted mutagenesis using the Cas protein rather than the nucleic acid encoding the Cas protein is advantageous because it avoids the introduction of exogenous DNA into the organism. Therefore, compositions containing the Cas protein and guide RNA can be used in the development of therapeutic drugs 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 eukaryotes, comprising a guide RNA or DNA encoding a guide RNA specific to target DNA, and a Cas protein-coding nucleic acid or Cas protein. In addition, the present invention provides the use of a composition for inducing targeted mutagenesis in eukaryotic cells or eukaryotes, comprising a guide RNA or DNA encoding a guide RNA specific to target DNA, and a Cas protein-coding nucleic acid or Cas protein. The guide RNA, Cas protein-coding nucleic acid, or Cas protein is as described above.

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

[0043] In another embodiment, the present invention provides a method for producing eukaryotic cells or eukaryotes having Cas protein and guide RNA, comprising the step of simultaneously or sequentially transfecting eukaryotic cells or eukaryotes with Cas protein-coding nucleic acid or Cas protein and guide RNA or DNA encoding guide RNA. The guide RNA, Cas protein-coding nucleic acid, or Cas protein is as described above.

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

[0045] In another embodiment, the present invention provides a eukaryotic cell or eukaryote containing a guide RNA specific to a target DNA or DNA encoding a guide RNA, and a Cas protein-coding nucleic acid or Cas protein. Eukaryotic cells or eukaryotes can be created by transferring a composition containing a guide RNA or DNA encoding a guide RNA specific to a target DNA, and a Cas protein-coding nucleic acid or Cas protein into a cell or organism. Eukaryotic cells may be, but are not limited to, yeast, fungi, protists, higher plants, and insect or amphibian cells, as are commonly used in the art, or mammalian cells such as CHO, HeLa, HEK293, and COS-1, e.g., cultured cells (in vitro), transplanted cells and primary cultured cells (in vitro and ex vivo), and in vivo cells, as well as mammalian cells such as human cells. Furthermore, organisms may be yeast, fungi, protists, plants, higher plants, insects, amphibians, or mammals.

[0046] In another embodiment of the present invention, the present invention provides a method for cleaving target DNA or inducing targeted mutagenesis in eukaryotic cells or eukaryotes, comprising the step of treating cells or organisms having target DNA with a composition containing a guide RNA or DNA encoding the guide RNA specific to the target DNA, and a Cas protein-coding nucleic acid or Cas protein. The step of treating cells or organisms with the composition may be carried out by transferring the composition, which contains a guide RNA or DNA encoding the guide RNA specific to the target DNA, and a Cas protein-coding nucleic acid or Cas protein, into the cells or organism. As described above, such transfers can be carried out by microinjection, transfection, electroporation, etc.

[0047] In another aspect of the present invention, the present invention provides an embryo having a genome edited with the RGEN composition containing a guide RNA or DNA encoding a guide RNA specific to a target DNA, and a Cas protein-coding nucleic acid or Cas protein. Any embryo can be used in the present invention, and for the present invention, the embryo may be a mouse embryo. Embryos are produced by injecting PMSG (mare serum gonadotropin) and hCG (human chorionic gonadotropin) into female mice between 4 and 7 weeks of age, and the superovulating female mice can be mated with males, and the fertilized embryos can be collected from the fallopian tubes. The RGEN composition introduced into the embryo can cleave the target DNA complementary to the guide RNA through the action of the Cas protein, thereby inducing mutations in the target DNA. Therefore, embryos into which this RGEN composition has been introduced have an edited genome.

[0048] In one particular embodiment, it was found that the RGEN composition could induce mutations in mouse embryos, and these mutations could be transmitted to offspring. The method for introducing the RGEN composition into the embryo may be any method known in the art, such as microinjection, stem cell insertion, or retrovirus insertion. Preferably, microinjection may be used.

[0049] In another aspect, the present invention provides a genome-modified animal obtained by transferring an embryo having a genome edited with the RGEN composition into the fallopian tube 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 animal is not limited. Genome-modified animals possess mutations resulting from targeted mutagenesis based on this RGEN composition. These mutations can be any one of the following: deletion, insertion, translocation, or inversion. The site of the mutation is determined by the sequence of the guide RNA in the RGEN composition. Genome-modified animals with gene mutations can be used to determine the function of those genes.

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

[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 aspect 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 Cas protein specific to a target DNA sequence. In addition, the present invention provides a composition for genotyping nucleic acid sequences of pathogenic microorganisms in an isolated biological sample, comprising a guide RNA and Cas protein specific to a target DNA sequence. The guide RNA, Cas protein-coding nucleic acid, or Cas protein is as described above.

[0052] As used herein, the term “genotyping” refers to “restriction fragment length polymorphism (RFLP) assay.” RFLP can be used for 1) detecting indels in cells or organisms induced by artificial nucleases, 2) determining the genotype of spontaneous mutations or polymorphisms in cells or organisms, or 3) determining the genotype of the DNA of infectious pathogenic microorganisms, including viruses or bacteria. Mutations or polymorphisms can be induced in cells by artificial nucleases. Artificial nucleases may be, but are not limited to, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), or RGENs. As used herein, the term “biological sample” includes, but is not limited to, analytical samples such as tissues, cells, whole blood, serum (semm), plasma, saliva, sputum, cerebrospinal fluid, or urine.

[0053] Mutations or polymorphisms can be spontaneous mutations or polymorphisms. Mutations or polymorphisms are induced by pathogenic microorganisms. That is, if pathogenic microorganisms are detected and it is confirmed that a biological sample is infected, then mutations or polymorphisms result from infection by the pathogenic microorganisms. Pathogenic microorganisms may be viruses or bacteria, but are not limited to them.

[0054] Artificial nuclease-induced mutations are detected by various methods, including mismatch-sensitive Surveyor or T7 endonuclease I (T7E1) assays, RFLP analysis, fluorescence PCR, DNA lysis analysis, and Sanger sequencing and deep sequencing. While T7E1 and Surveyor assays are widely used, they detect heteroduplexes (formed by hybridization of a mutant sequence with a wild-type sequence or two different mutant sequences); they often underestimate mutation frequencies because they cannot detect homoduplexes formed by hybridization of two identical mutant sequences. Consequently, these assays cannot distinguish between homozygous biallele mutant clones and wild-type cells, nor can they distinguish between heterozygous biallele mutants and heterozygous monoallele mutants (Figure 22). In addition, sequence polymorphisms near the nuclease target site can lead to confounding results because the enzyme can cleave the heteroduplex formed by the hybridization of these different wild-type alleles. RFLP analysis is preferred because 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 aspect 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 nucleic acid sequences of pathogenic microorganisms in an isolated biological sample, comprising a guide RNA and Cas protein specific to a target DNA sequence. The guide RNA, Cas protein-coding nucleic acid, or Cas protein is 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 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, Cas protein-coding nucleic acid, or Cas protein is as described above.

[0057] (Examples) The present invention will be described in further detail below 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 assay 1-1. DNA cleavage activity of the Cas9 protein First, the DNA cleavage activity of Cas9 derived from Streptococcus pyogenes was tested in vitro, both in the presence and absence of chimeric guide RNA. For this purpose, a recombinant Cas9 protein expressed in E. coli and purified was used to cleave pre-digested plasmid DNA or circular plasmid DNA containing a 23-base pair (bp) human CCR5 target sequence. The Cas9 target sequence consists of a 20-bp DNA sequence complementary to the crRNA or chimeric guide RNA and a trinucleotide (5'-NGG-3') protospacer flanking motif (PAM) recognized by Cas9 itself (Figure 1a).

[0059] Specifically, the Cas9 coding sequence (4,104 bp) derived from the Streptococcus pyogenes strain M1 GAS (NC_002737.1) was reconstructed using a human codon usage table and synthesized using oligonucleotides. First, a 1-kb DNA segment was 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 using four 1-kbp DNA segments by overlap PCR. 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 the HA epitope and nuclear localization signal (NLS) was added to the C-terminus of Cas9. The expression and nuclear localization of the Cas9 protein in HEK 293T cells were confirmed by Western blotting using an anti-HA antibody (Santa Cruz).

[0060] Subsequently, the Cas9 cassette was subcloned into pET28-b(+) and transformed into BL21(DE3). Cas9 expression was induced using 0.5 mM IPTG at 25°C for 4 hours. The Cas9 protein with a His6-tag at the C-terminus 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). The purified Cas9 (50 nM) was incubated with supercoiled plasmid DNA or pre-digested plasmid DNA (300 ng) and chimeric RNA (50 nM) in 20 μl reaction volume NEB buffer 3 at 37°C for 1 hour. The digested DNA was analyzed by electrophoresis using a 0.8% agarose gel. Cas9 efficiently cleaved plasmid DNA at the expected location only in the presence of synthetic RNA, and did not cleave control plasmids that lacked a target sequence (Figure 1b).

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

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

[0063] On the other hand, in vitro transcribed chimeric RNA was prepared as follows. Using the MEGAshortscript T7 kit (Ambion), RNA was transcribed in vitro by a run-off reaction according to the manufacturer's instructions. The template for in vitro transcription of RNA was generated by annealing two complementary single-stranded DNAs or by PCR amplification (Table 1). The transcribed RNA was separated on an 8% denatured urea-PAGE gel. Gel sections containing RNA were excised and transferred to probe 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 spectroscopic analysis.

[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. GFP-expressing cells were found to be obtained only when cells were first transfected with a Cas9 plasmid and then 12 hours later with a guide RNA (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 with Cas9 plasmid and guide RNA rather than by simultaneous transfection. [Table 1]

[0065] 1-3. Targeted disruption 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, we used the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza), following the manufacturer's protocol, with 2 × 10⁶ cells. 6 K562 cells were transfected with 20 μg of Cas9-coding 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 hours, 10-40 μg of in vitro transcribed chimeric RNA was used, 1 × 10⁶ 6 K562 cells were nucleofected. In vitro transcribed chimeric RNA was prepared as described in Examples 1-2. Cells were harvested two days after RNA transfection, and genomic DNA was isolated. The region containing the target site was PCR amplified using the primers listed in Table 1. The amplicons were subjected to the T7E1 assay as previously described (3). For sequencing analysis, the PCR product corresponding to the genome modification was purified and cloned into a T-Blunt vector using the T-Blunt PCR Cloning Kit (SolGent). The cloned product was sequenced using M13 primers.

[0067] Mutations were found to be induced only when cells were sequentially transfected with a Cas9 coding plasmid, followed by guide RNA (Figure 3). The mutation frequency estimated from relative DNA band intensity (Indels (%) in Figure 3a) was RNA dose-dependent, ranging from 1.3% to 5.1%. DNA sequencing analysis of PCR amplicons confirmed the induction of RGEN-mediated mutations at endogenous sites. Indels and microhomologies, characteristic of error-prone NHEJ, were observed at the target sites. 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). Inducing mutations in cells previously required sequential transfection with both the Cas9 plasmid and the guide RNA. However, with the guide RNA-coding plasmid, sequential transfection was unnecessary, and cells were simultaneously transfected with both the Cas9 plasmid and the guide RNA-coding plasmid.

[0068] On the other hand, both ZFNs and TALENs have been successfully developed to disrupt the human CCR5 gene, which encodes the G protein-coupled chemokine receptor, an essential co-receptor for HIV infection (3-6). CCR5-specific ZFNs are currently in clinical trials in the United States for the treatment of AIDS (7). However, these ZFNs and TALENs have off-target effects, which include both local mutations at sites homologous to the on-target sequence (6, 8-10), as well as genomic rearrangements resulting from the repair of two simultaneously occurring DSBs induced at on-target and off-target sites (11-12). The most prominent off-target site associated with these CCR5-specific artificial nucleases is located at the CCR2 locus, a close homolog of CCR5, situated 15-kbp upstream of CCR5. To avoid off-target mutations in the CCR2 gene, as well as undesirable deletions, inversions, and duplications of the 15-kbp chromosomal segment between the CCR5 on-target site and the CCR2 off-target site, we have deliberately selected the target site of our CCR5-specific RGEN to recognize a region within the CCR5 sequence that does not have apparent homology to the CCR2 sequence.

[0069] The inventors 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 nearly homologous to the target 23-bp sequence. As expected, no such sites were found in the CCR2 gene. Instead, four sites were identified, each with a 3-base mismatch with the on-target site (Figure 4a). The T7E1 assay showed no mutations detected at these sites (assay sensitivity, ~0.5%), demonstrating the excellent specificity of RGEN (Figure 4b). Furthermore, PCR was used to detect the induction of chromosomal deletions in cells separately transfected with plasmids encoding CCR5-specific ZFN and RGEN. ZFN induced deletions, but RGEN did not (Figure 4c).

[0070] Next, the RGEN was reprogrammed by replacing the CCR5-specific guide RNA with a novel synthetic RNA designed to target the human C4BPB gene, which encodes the β-chain of the transcription factor C4b-binding protein. This RGEN induced mutations at a high frequency at the chromosomal target site in K562 cells (Figure 3b). Mutation frequencies, measured by 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 precisely at the cleavage site, a pattern also observed at the CCR5 target site. These results demonstrate that the RGEN cleaves the target DNA on the chromosome at the expected location within the cell. [Examples]

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

[0072] Here, we induced targeted disruption of endogenous genes in human cells using recombinant Cas9 protein complexed with in vitro transcribed guide RNA. Recombinant Cas9 protein fused with a hexahistidine tag was expressed in E. coli and purified using standard Ni-ion affinity chromatography and gel filtration. The purified recombinant Cas9 protein was concentrated in a storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol). The Cas9 protein / sgRNA complex was directly introduced into K562 cells by nucleofection. Specifically, using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza), and following the manufacturer's protocol, 22.5-225 (1.4-14 μM) of 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 mixed with 1 × 10⁶ of Cas9 protein. 6 K562 cells were transfected. After nucleofection, the cells were placed in growth medium in a 6-well plate and incubated for 48 hours. 2 × 10 5 When K562 cells were transfected using a method scaled down to 1 / 5, nucleofect was performed in 20 μl of solution with 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). Nucleofected cells were then placed in growth medium in 48-well plates. After 48 hours, cells were harvested and genomic DNA was isolated. Genomic DNA regions across target sites were amplified by PCR and subjected to the T7E1 assay.

[0073] As shown in Figure 10, the Cas9 protein / sgRNA complex induced targeted mutations at the CCR5 locus at a dose-dependent frequency of 4.8–38% for sgRNA or Cas9 protein, which was comparable to the frequency obtained by transfection with the Cas9 plasmid (45%). The Cas9 protein / crRNA / tracrRNA complex was able to induce mutations at a frequency of 9.4%. Cas9 protein alone could not induce mutations. 2 × 10 5 When cells were transfected with Cas9 protein and sgRNA at a 1 / 5 scaled-down dose, the mutation frequency at the CCR5 locus ranged from 2.7% to 57% in a dose-dependent manner, which was higher than the frequency obtained by simultaneous transfection with Cas9 plasmid and sgRNA plasmid (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-induced genome editing in mice To investigate the gene targeting ability of RGEN in pronuclear (PN) stage mouse embryos, we used the forkhead box N1 (Foxn1) gene, which is important for thymic 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). To evaluate the genome editing activity of Foxn1-RGEN, we injected Cas9 mRNA (10-ng / μl solution) along with various doses of sgRNA into the cytoplasm of PN-stage mouse embryos (Figure 5a), and performed a T7 endonuclease I (T7E1) assay (Kim et al. 2009) using genomic DNA obtained from the in vitro cultured embryos (Figure 6a).

[0076] Alternatively, we directly injected RGEN into the cytoplasm or pronucleus of single-cell mouse embryos in the form of recombinant Cas9 protein (0.3–30 ng / μl) complexed with a 2x molar excess of Foxn1-specific sgRNA (0.14–14 ng / μl), and analyzed Foxn1 gene mutations in embryos cultured in vitro (Figure 7). Specifically, Cas9 mRNA and sgRNA were synthesized in vitro from linear DNA templates using the 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 diethyl pyrocarbonate (DEPC, Sigma) treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4). Templates for sgRNA synthesis were generated using oligonucleotides listed in Table 3. Recombinant Cas9 protein was obtained from ToolGen, Inc. [Table 3]

[0077] All animal experiments were conducted in accordance with the Korea Food and Drug Administration (KFDA) guidelines. The protocols were reviewed and approved by the Animal Experiment Committee (IACUC) of the Laboratory Animal Research Facility at Yonsei University (Approval No.: 2013-0099). All mice were maintained in a 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 of pregnant mare serum gonadotropin (PMSG, Sigma) and 5 IU of human chorionic gonadotropin (hCG, Sigma) at 48-hour intervals. The superovulated female mice were mated with FVB / NTac breeding males, and fertilized embryos were recovered from the oviducts. Using a piezo-driven micromanipulator (Prime Tech), Cas9 mRNA and sgRNA from 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 to the fallopian tubes of pseudo-pregnant surrogate mothers to create live animals, or cultured in vitro for further analysis.

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

[0080] The 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 standard PCR genotyping of F1 offspring, the following primer pairs were used for both the 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 proportion of mutants (number of mutant embryos / total number of 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, but most mutations were small deletions (Figure 6c), similar to mutations induced by ZFNs and TALENs (Kim et al., 2013). In the case of Cas9 protein injection, these injection doses and methods did not significantly affect the in vitro survival and development of mouse embryos, with over 70% of RGEN-injected embryos hatching normally in both experiments. Furthermore, the percentage of mutants obtained by Cas9 protein injection was dose-dependent, reaching 88% with the highest dose via pronuclear injection and 71% with intracytoplasmic injection (Figures 7a and 7b). Similar to the mutation patterns induced by Cas9 mRNA and sgRNA (Figure 6c), mutations induced by the Cas9 protein-sgRNA complex were mainly small deletions (Figure 7c). These results clearly demonstrate that RGEN has high gene targeting activity in mouse embryos.

[0082] Driven by the high mutation rate and low cytotoxicity induced by RGEN, we created living animals by transferring mouse embryos into the fallopian tubes of pseudopregnant surrogate mothers. Notably, the birth rate was very high, ranging from 58% to 73%, and was not affected by the increase in Foxn1-sgRNA dose (Table 6). [Table 6]

[0083] Of the 147 newborns, we obtained 99 mutant founder mice. Consistent with the results observed in cultured embryos (Figure 6b), the mutant rate was proportional to the dose of Foxn1-sgRNA, reaching a maximum of 93% (100 ng / μl Foxn1-sgRNA) (Tables 6 and 7, Figure 5b). [Table 7] TIFF0007855628000008.tif251148 TIFF0007855628000009.tif72151

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

[0085] To test germline transmission of the mutant allele, we crossed Foxn1 mutant founder #108, a mosaic with four different alleles (Figure 5c, and Table 8), with wild-type mice and observed the genotypes of the F1 offspring. [Table 8] TIFF0007855628000011.tif54161

[0086] As expected, all offspring were heterozygous mutants possessing both the 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 reliably transmitted to the F1 offspring of animals. [Examples]

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

[0088] 4-2. Production of guide RNA To screen the genome sequence of the Arabidopsis gene encoding BRI1 for the presence of NGG motifs, also known as protospacer-adjacent motifs (PAMs), within exons required for Cas9 targeting, we identified two RGEN target sites within exons containing NGG motifs to disrupt the Arabidopsis BRI1 gene. sgRNAs were constructed in vitro using template DNA. Each template DNA was prepared by extension using two partially duplicated oligonucleotides (Macrogen, Table X1) and Phusion polymerase (Thermo Scientific) under the following conditions: 98°C for 30 seconds (98°C for 10 seconds, 54°C for 20 seconds, 72°C for 2 minutes) × 20, followed by 72°C for 5 minutes. [Table 9]

[0089] The extended DNA was purified and used as a template for in vitro production of guide RNA using the 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 two sgRNAs (11 μg / μl) were mixed in 20 μl of NEB3 buffer (New England Biolabs) and incubated at 37°C for 10 minutes.

[0090] 4-3. Transfection of the Cas9 / sgRNA complex into protoplasts Leaves of 4-week-old Arabidopsis seedlings, aseptically grown in Petri dishes, were digested in an enzyme solution (1% cellulase (cellulose) R10, 0.5% macerozyme R10, 450 mM mannitol, 20 mM MES pH 5.7, and CPW salt) at 25°C for 8–16 hours with shaking at 40 rpm in the dark. The enzyme / protoplast solution was filtered and centrifuged at 100 × g for 3–5 minutes. After counting cells using a hemocytometer under a microscope (×100), the protoplasts were resuspended in CPW solution. Finally, the protoplasts were hydrated in MMG solution (4 mM HEPES pH 5.7, 400 mM mannitol, and 15 mM MgCl2) at a rate of 1 × 10⁶ 6The protoplasts were resuspended in 1 / ml. To transfect the protoplasts with the Cas9 / sgRNA complex, 200 μL (200,000 protoplasts) of protoplast suspension was gently mixed in a 2 ml tube with 3.3 or 10 μL of Cas9 / sgRNA complex [Cas9 protein (6 μg / μL) and two sgRNAs (2.2 μg / μL each)] and 200 μL of 40% polyethylene glycol transfection buffer (40% PEG4000, 200 mM mannitol and 100 mM CaCl2). After incubation at room temperature for 5–20 minutes, transfection was stopped by adding a wash buffer containing W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl). Subsequently, the protoplasts were collected by centrifugation at 100×g for 5 minutes, washed with 1 ml of W5 solution, and centrifuged again at 100×g for 5 minutes. The density of the protoplasts was determined to be 1×10⁻⁶. 5 The solutions were adjusted to / ml and cultured in modified KM 8p liquid medium containing 400 mM glucose.

[0091] 4-4. Detection of mutations in Arabidopsis protoplasts and plants Protoplasts were collected 24 or 72 hours after transfection, and genomic DNA was isolated. Genomic DNA regions spanning two target sites were PCR amplified and subjected to the T7E1 assay. As shown in Figure 11, indels were induced by RGEN at a high frequency of 50%–70%. Surprisingly, 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). Plasmids were purified and subjected to Sanger sequencing using M13F primers. One mutant sequence had a 7-bp deletion at one site (Figure 12). The other three mutant sequences had a deletion of a ~220-bp DNA fragment between two RGEN sites. [Examples]

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

[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, 1% penicillin, and streptomycin.

[0094] 5-3. Expression and Purification of Cas9 Protein To express the Cas9 protein, E. coli BL21 cells were transformed with the Cas9-encoding pET28-(a) vector and inoculated onto Luria-Bertani (LB) agar medium containing 50 μg / mL kanamycin (Amresco, Solon, OH). The following day, single colonies were isolated and cultured overnight at 37°C in LB culture medium containing 50 μg / mL kanamycin. The next day, these seed cultures were inoculated into Luria culture medium containing 50 μg / mL kanamycin at 0.1 OD600 and incubated at 37°C for 2 hours 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, and the cells were cultured overnight at 30°C.

[0095] Cells were collected by centrifugation at 4000 rpm for 15-20 minutes, 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 cycle, 10-second pulse, 30-second pause, 10 minutes on ice). The soluble fraction was separated as supernatant after centrifugation at 15,000 rpm for 20 minutes at 4°C. Cas9 protein was purified at 4°C using a Ni-NTA agarose resin column (QIAGEN) and AKTA prime instrument (AKTA prime, GE Healthcare, UK). During this chromatography, the soluble protein fraction was added to the Ni-NTA agarose resin column (GE Healthcare, UK) at a flow rate of 1 mL / min. The column was washed with a washing buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole, 1× protease inhibitor cocktail), and the bound protein was eluted at a flow rate of 0.5 ml / min using an elution buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 250 mM imidazole, 1× protease inhibitor cocktail). The pooled eluted fraction was concentrated and dialyzed against a 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 the Bradford assay (Biorad, Hercules, CA), and purity was analyzed by SDS-PAGE using bovine serum albumin as a control.

[0096] 5-4. Conjugate formation 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 of DW were gently mixed using a rotor at room temperature for 2 hours and overnight at 4°C. To remove unconjugated maleimide-9R4L, the samples were dialyzed against DPBS (pH 7.4) at 4°C for 24 hours using a 50 kDa molecular weight cutoff membrane. The Cas9-9R4L protein was recovered from the dialyzed 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 varying amounts of C9R4LC peptide (ranging in weight ratios from 1 to 40) in 100 μl of DPBS (pH 7.4). This mixture was incubated at room temperature for 30 minutes and then diluted 10-fold with deionized water that did not contain RNAse. 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. Treatment of Cas9 protein and sgRNA Cells were treated with Cas9-9R4L and sgRNA-C9R4LC as follows: 1 μg of sgRNA and 15 μg of C9R4LC peptide were added to 250 mL of OPTIMEM medium and incubated at room temperature for 30 minutes. 24 hours after seeding, the cells were washed with OPTIMEM medium and treated with the sgRNA-C9R4LC complex at 37°C for 4 hours. The cells were washed again with OPTIMEM medium and treated with Cas9-9R4L at 37°C for 2 hours. After treatment, the medium was replaced with serum-containing complete medium and incubated at 37°C for 24 hours before the next treatment. The same procedure was repeated for 3 consecutive days for multiple treatments with Cas9 and sgRNA.

[0099] 5-7. Cas9-9R4L and sgRNA-9R4L can edit endogenous genes in cultured mammalian cells without the use of further delivery methods. To determine whether Cas9-9R4L and sgRNA-9R4L can edit endogenous genes in cultured mammalian cells without the use of further delivery methods, we treated 293 cells with Cas9-9R4L and sgRNA-9R4L targeting the CCR5 gene, and analyzed their genomic DNA. The T7E1 assay showed that 9% of the CCR5 gene was disrupted in cells treated with both Cas9-9R4L and sgRNA-9R4L, while no CCR5 gene disruption was observed in control cells, including untreated cells, cells treated with either Cas9-9R or sgRNA-9R4L, or cells treated with both unmodified Cas-9 and sgRNA (Figure 13). This suggests that treatment with Cas9-9R4L protein and sgRNA conjugated with 9R4L, rather than unmodified Cas-9 and sgRNA, can induce 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 differ from the on-target site by 3 to 5 nucleotides. However, we noticed some differences between our RGENs and those used by other groups. First, we used dual RNAs of crRNA and tracrRNA instead of single-stranded guide RNA (sgRNA) composed of the essential parts of crRNA and tracrRNA. Second, we transfected K562 cells (not HeLa cells) with synthetic crRNA instead of the plasmid encoding crRNA. HeLa cells were transfected with the crRNA-encoding plasmid. Other groups used the sgRNA-encoding plasmid. Third, our guide RNAs have two additional guanine nucleotides at the 5' end, which are required for efficient in vitro transcription by T7 polymerase. Such additional nucleotides were not included in the sgRNAs used by other groups. Therefore, the RNA sequence of our guide RNA can be shown as 5'-GGX 20 while, on the other hand, 5'-GX 19 represents the sequences used by other groups, and X 20 or GX 19This corresponds to a 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, in K562 cells, we measured the mutation frequencies at on-target and off-target sites by the T7E1 assay, comparing our method using in vitro transcribed dual RNA with a method transfecting with an sgRNA coding plasmid. 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 an indel at one effective off-target site (called OT1-11, Figure 14) that differed by 3 nucleotides from the on-target site when using synthetic dual RNA. However, the synthetic dual RNA did not identify the other effective off-target sites (OT1-3) that differed by 2 nucleotides from the on-target site.

[0101] Next, we investigated whether the addition of two guanine nucleotides to the 5' end of sgRNA could make RGEN more specific, using 5'-GGX. 20 (or 5'-GGGX) 19 ) sgRNA and 5'-GX 19 The analysis was performed by comparing it with sgRNA. Four GX complexes formed with Cas9. 19 sgRNAs efficiently induced indels at both on-target and off-target sites, tolerating up to 4 nucleotide mismatches. In stark contrast, GGX 20 sgRNA effectively identified off-target sites. In fact, we found four GGX 20 When using sgRNA, the T7E1 assay detected very few RGEN-induced indels at 6 of the 7 effective off-target sites (Figure 15). However, we found that two GGX 20sgRNA (VEGFA sites 1 and 3) corresponds to GX 19 We noticed that the activity at the on-target site was lower than that of sgRNA. These results suggest that the additional nucleotides at the 5' end may affect the mutation frequency at both the on-target and off-target sites, possibly by altering the stability, concentration, or secondary structure of the guide RNA. These results are attributed to three factors: the use of synthetic guide RNA instead of guide RNA coding plasmids, the use of dual RNA instead of sgRNA, and GX 19 GGX, not sgRNA 20 This suggests that the use of sgRNA has a cumulative effect on the identification of off-target sites. [Examples]

[0102] Paired Cas9 Knickers In principle, single-strand breaks (SSBs) cannot be repaired by error-prone NHEJs, but they trigger highly fidelity homologous recombination repair (HDR) or base excision repair. However, nickase-induced targeted mutagenesis by HDR is far less efficient than nuclease-induced mutagenesis. We hypothesized that paired Cas9 nickase would generate complex DSBs that trigger DNA repair by NHEJ or HDR, leading to efficient mutagenesis (Figure 16a). Furthermore, paired nickase would double the specificity of Cas9-mediated genome editing.

[0103] We first tested several Cas9 nucleases and nickases designed to target the AAVS1 locus (Figure 16b) in vitro by fluorescent capillary electrophoresis. Unlike Cas9 nucleases that cleaved both strands of the DNA substrate, Cas9 nickases composed of a Cas9 mutant (D10A Cas9) in which the guide RNA and catalytic aspartate residue were changed to alanine cleaved only one strand, producing site-specific nicks (Figures 16c, d). Interestingly, however, some nickases (AS1, AS2, AS3, and S6 in Figure 17a) 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 created two adjacent nicks on opposing DNA strands produced indels at frequencies of 14%–91%, comparable to the effect of paired nucleases (Figure 17a). Repair of two nicks that produced 5' overhangs led to indel formation at a much higher frequency than nicks that produced 3' overhangs at three genomic loci (Figures 17a and 18). In addition, paired nicksase enabled targeted genome editing via homologous recombination repair more efficiently than single nicksase (Figure 19).

[0104] We then measured the mutation frequencies at off-target sites of paired nickase and nuclease using deep sequencing. Cas9 nucleases complexed with three sgRNAs induced off-target mutations at six sites differing by one or two nucleotides from their corresponding on-target sites, at 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, at the first position in PAM (i.e., N in NGG), which differs by one nucleotide from its on-target site, can be considered another on-target site. As expected, Cas9 nucleases complexed with S2 sgRNA were equally effective at this site and the on-target site. In stark contrast, D10A Cas9, when complexed with S2 sgRNA and AS2 sgRNA, distinguished this site from the on-target site by a 270-fold difference. This paired nickase also distinguished the AS2 off-target sites (Off-1 and Off-9 in Figure 17b) from the on-target site by 160-fold and 990-fold differences, respectively. [Examples]

[0105] Chromosomal DNA splicing induced by paired Cas9 nickase It has been reported that two simultaneously occurring double-segment breaks (DSBs) induced by artificial nucleases such as ZFNs and TALENs can promote large deletions of intervening chromosomal segments. We investigated whether two single-segment breaks (SSBs) induced by paired Cas9 nicks can also induce deletions in human cells. We detected deletion events using PCR and found that seven paired nicks, similar to paired Cas9 nucleases, efficiently induced deletions of chromosomal segments up to 1.1-kbp (Figure 20a, b). Deletion events were confirmed by DNA sequencing of the PCR products (Figure 20c). Interestingly, sequences matching sgRNA were perfectly preserved in two of the seven deletion-specific PCR amplicons (underlined in Figure 20c). In contrast, the Cas9 nuclease pair did not produce sequences containing the complete target site. This finding suggests that the two distant nicks were not converted into two independent DSBs that promoted the deletion of the intervening chromosomal segment. Furthermore, because its melting temperature is very high, it is unlikely that two nicks separated by more than 100 bp could produce a complex double-segment bridge (DSB) with a large protrusion under physiological conditions.

[0106] We propose that two distant nicks are repaired by strand substitution in a head-to-head direction, leading to the formation of a double-sided spinal block (DSB) in the center, and that this repair by NHEJ results in a small deletion (Figure 20d). During this process, the two target sites are perfectly preserved, so nickase again generates an SSB, repeating this cycle until the target sites are deleted. This mechanism explains why two offset nicks that generate 5' overhangs, rather than a nick that generates a 3' overhang, efficiently induced indels at three loci.

[0107] We then investigated whether Cas9 nucleases and nickases could induce undesirable chromosomal translocations resulting from NHEJ repair of on-target and off-target DNA breaks (Figure 21a). We were able to detect Cas9 nuclease-induced translocations using PCR (Figures 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 (Figure 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 induce indels at off-target sites where their corresponding nucleases induce mutations. Furthermore, unlike nucleases, paired nickases did not promote undesirable translocations associated with off-target DNA cleavage. In principle, paired nickases double the specificity of Cas9-mediated mutagenesis and expand the utility of RNA-inducible enzymes in applications requiring precise genome editing, such as gene therapy and cell therapy. One caveat to this approach is that it requires two highly active sgRNAs to construct an efficient nickase pair, limiting the targetable sites. Not all sgRNAs are equally active, as demonstrated in this study and others. When a single clone, rather than a population of cells, is used for further research or applications, selecting and optimizing guide RNAs that exhibit specific sequences in the genome may be sufficient to avoid off-target mutations associated with Cas9 nucleases. We propose that both Cas9 nucleases and paired nickases are powerful options for facilitating precise genome editing in cells and organisms. [Examples]

[0109] Genotyping using RNA-induced endonucleases derived from CRISPR / Cas Next, we hypothesized that RGEN could be used in place of conventional restriction enzymes for restriction fragment length polymorphism (RFLP) analysis. Artificial nucleases, including RGEN, induce indels at target sites when the double-segment breaks (DSBs) induced by the nuclease are repaired by the error-prone non-homologous end-joining (NHEJ) system. Designed to recognize target sequences, RGEN would not be able to cleave mutant sequences containing indels, but would effectively cleave 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% denatured urea-PAGE gel. Gel sections containing the RNA were excised and transferred to elution buffer. The RNA was recovered in water without nucleases, followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. The purified RNA was quantified by spectroscopic analysis. The template for crRNA was a sequence with the value 5'-GAAATTAATACGACTCACTATAGGX 20 GTTTTAGAGCTATGCTGTTTTG-3'(Sequence ID 76)(X 20 The target sequence (is shown as oligonucleotide) was prepared by annealing it with its complementary oligonucleotide. The template for tracrRNA was prepared using Phusion polymerase (New England Biolabs) and forward and reverse oligonucleotides. (5'-GAAATTAATACGACTCACTATAGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCG-3' (Sequence ID 77) and It was synthesized by elongation of 5'-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATG-3' (SEQ ID NO: 78)).

[0111] 9-2. Purification of Recombinant Cas9 Protein A Cas9 DNA construct encoding Cas9 fused with a His6-tag at the C-terminus, as used in our previous example, was inserted into a pET-28a expression vector. Recombinant Cas9 protein was expressed in E. coli strain BL21(DE3) cultured in LB medium after induction with 1 mM IPTG at 25°C for 4 hours. 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. 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 described below. Briefly, PCR products amplified using genomic DNA were denatured at 95°C, re-annealed at 16°C, and incubated with 5 units of T7 endonuclease I (New England BioLabs) at 37°C for 20 minutes. The reaction products were separated using 2-2.5% agarose gel electrophoresis.

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

[0115] 9-6. RFLP Strategy Novel RGENs with desired DNA specificity can be easily constructed by replacing crRNA, and once the recombinant Cas9 protein is obtained, de novo purification of the custom protein is not required. Artificial nucleases containing RGENs induce small insertions or deletions (indels) at target sites when the double-segment breaks (DSBs) induced by the nuclease are repaired by error-prone non-homologous end joinings (NHEJs). RGENs designed to recognize target sequences efficiently cleave wild-type sequences but cannot cleave mutant sequences containing indels (Figure 22).

[0116] We first tested whether RGEN could selectively cleave plasmids containing wild-type or modified C4BPB target sequences with 1-3 nucleotide indels at the cleavage site. None of the six plasmids with these indels were cleaved by C4BPB-specific RGEN5, which consists of target-specific crRNA, tracrRNA, and recombinant Cas9 protein (Figure 23). In contrast, plasmids with complete target sequences 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 detecting mutations induced by the same RGEN, we used genetically modified K562 human cancer cell clones established using an RGEN targeting the C4BPB gene (Table 12). [Table 12]

[0118] The C4BPB mutant clones used in this study exhibited a variety of mutations, ranging from 94 bp deletions to 67 bp insertions (Figure 24a). Importantly, all mutations in the mutant clones resulted in a lack of the RGEN target site. Of the six C4BPB clones analyzed, four possessed both the wild-type and mutant alleles (+ / -), while two possessed only the mutant allele (- / -).

[0119] PCR products across RGEN target sites amplified from wild-type K562 genomic DNA were completely digested by RGEN, which consists of target-specific crRNA, tracrRNA, and purified recombinant Cas9 protein expressed in E. coli (Figure 24b / lane 1). When C4BPB mutant clones were subjected to RFLP analysis using RGEN, PCR amplicons of + / - clones possessing both wild-type and mutant alleles were partially digested, while amplicons of - / - clones lacking the wild-type allele were not digested at all, and did not produce cleavage products corresponding to the wild-type sequence (Figure 24b). Even a single nucleotide insertion at the target site prevented digestion of the amplified mutant allele by C4BPB RGEN (clones #12 and #28), demonstrating high specificity of RGEN-mediated RFLP. We also subjected the PCR amplicons to a mismatch-sensitive T7E1 assay (Figure 24b). Notably, the T7E1 assay could not distinguish - / - clones from + / - clones. To make matters worse, because annealing of the same mutant sequence forms a homozygous double helix, the T7E1 assay cannot distinguish homozygous mutant clones with the same mutant sequence from wild-type clones. Therefore, RGEN-mediated RFLP has a significant advantage over conventional mismatch-sensitive nuclease assays in the analysis of mutant clones induced by artificial nucleases such as ZFN, TALEN, and RGEN.

[0120] 9-8. Quantitative assay of 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 did not correlate well with the mutation frequency estimated from the ratio, and were particularly inaccurate at high mutant percentages where complementary mutant sequences could hybridize 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) to the analysis of mutant mouse founders established by TALEN injection into mouse single-cell embryos (Figure 26a). We designed and used an RGEN that recognizes the TALEN target site in the Pibf1 gene (Table 10). Genomic DNA was isolated from wild-type and mutant mice, amplified by PCR, and then subjected to RGEN genotyping. RGEN genotyping successfully detected a variety of mutations ranging from 1 to 27 bp deletions (Figure 26b). Unlike the T7E1 assay, RGEN genotyping enabled the discriminative detection of + / - and - / - founders.

[0122] 9-10. Detection of CCR5-specific ZFN-induced mutations in human cells using RGEN In addition, we used RGEN to detect mutations induced in human cells by yet another type of artificial nuclease, the CCR5-specific ZFN (Figure 27). These results demonstrate that RGEN can detect mutations induced by nucleases other than RGEN itself. In fact, we expect that RGEN could be designed to detect most, though not all, mutations induced by artificial nucleases. The only constraint in designing the RGEN genotyping assay is the need for a GG or AG (CC or CT in the complementary strand) dinucleotide in the PAM sequence recognized by the Cas9 protein, which appears on average once every 4 bp. Indels induced anywhere within the seed region of a few bases in the crRNA and PAM nucleotides are expected to interfere with RGEN catalytic DNA cleavage. Indeed, we identified 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 novel 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 adjacent to the target site (Figure 25c). However, as expected, the same RGEN used for gene disruption completely cleaved PCR products from wild-type cells but partially cleaved PCR products from RGEN-transfected cells, indicating the presence of RGEN-induced indels at the target site. This result demonstrates that RGEN-RFLP analysis has clear advantages over the T7E1 assay, particularly when it is unclear whether the target gene has polymorphism or diversity in the target cells.

[0124] 9-12. Detection of recurrent mutations and spontaneous polymorphisms in cancer by RGEN-RFLP analysisRGEN-RFLP analysis has applications beyond genotyping of induced nuclease-induced mutations. We attempted to detect recurrent mutations and spontaneous polymorphisms observed in cancer using RGEN genotyping. We selected the human colorectal cancer cell line HCT116, which has a gain-of-function 3-bp deletion in the oncogenic CTNNB1 gene encoding β-catenin. PCR products amplified from HCT116 genomic DNA were partially cleaved by both wild-type-specific RGEN and mutation-specific RGEN, consistent with heterozygous genotypes in HCT116 cells (Figure 29a). Clearly in contrast, PCR products amplified from DNA derived from HeLa cells containing only the wild-type allele were completely digested by wild-type-specific RGEN and not cleaved at all by mutation-specific RGEN.

[0125] We also noted that HEK293 cells have a 32-bp deletion (del32) in the CCR5 gene, which encodes a co-receptor essential for HIV infection, and that homozygous del32 CCR5 carriers are immune to HIV infection. We designed one RGEN specific to the del32 allele and another RGEN against the wild-type allele. As expected, the wild-type specific RGEN completely cleaved PCR products obtained from K562, SKBR3, or HeLa cells (used as wild-type controls), but partially cleaved PCR products from HEK293 cells (Figure 30a), confirming the presence of an uncleavable del32 allele in HEK293 cells. Unexpectedly, however, the del32 specific RGEN efficiently cleaved PCR products from wild-type cells as well as those from HEK293 cells. Interestingly, this RGEN had an off-target site with a single nucleotide mismatch immediately downstream of the on-target site (Figure 30). These results suggest that while RGEN can detect spontaneously occurring indels, their off-target effects prevent them from distinguishing sequences containing single nucleotide polymorphisms or point mutations.

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

[0127] The data presented above propose RGEN as a foundation for using simple and reliable RFLP analysis for various sequence mutations. Due to its high flexibility in reprogramming target sequences, RGEN can be used to detect a wide range of genetic mutations (single nucleotide mutations, small insertions / deletions, structural mutations), including disease-associated recurrent mutations, genotypes related to patient drug response, and mutations induced by artificial nucleases in cells. In this specification, we used RGEN genotyping to detect mutations induced by artificial nucleases in cells and animals. In principle, RGEN can also be used to specifically detect and cleave spontaneous polymorphisms and mutations.

[0128] Based on the above description, it will be understood by those skilled in the art that various alternatives to the embodiments of the present invention described herein may be used in carrying out the invention without departing from the technical idea or essential features of the invention as defined in the claims below. In this regard, the above-described embodiments are for illustrative purposes only, and the invention is not intended to be limited by these embodiments. The scope of the invention should be understood to include all modifications or variations derived from the intent and scope of the claims below 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) JPEG0007855628000016.jpg245137 JPEG0007855628000017.jpg244135 JPEG0007855628000018.jpg244145 JPEG0007855628000019.jpg245140 JPEG0007855628000020.jpg243136 JPEG0007855628000021.jpg245144 JPEG0007855628000022.jpg244145 JPEG0007855628000023.jpg245153 JPEG0007855628000024.jpg243143 JPEG0007855628000025.jpg244146 JPEG0007855628000026.jpg244142 JPEG0007855628000027.jpg245142 JPEG0007855628000028.jpg244137 JPEG0007855628000029.jpg244158 JPEG0007855628000030.jpg244148 JPEG0007855628000031.jpg245144 JPEG0007855628000032.jpg243146 JPEG0007855628000033.jpg244148 JPEG0007855628000034.jpg245139 JPEG0007855628000035.jpg244150 JPEG0007855628000036.jpg243149 JPEG0007855628000037.jpg245146 JPEG0007855628000038.jpg244145 JPEG0007855628000039.jpg243135 JPEG0007855628000040.jpg245143 JPEG0007855628000041.jpg244138 JPEG0007855628000042.jpg245134 JPEG0007855628000043.jpg244125 JPEG0007855628000044.jpg243142 JPEG0007855628000045.jpg244134 JPEG0007855628000046.jpg244138 JPEG0007855628000047.jpg244130 JPEG0007855628000048.jpg244132 JPEG0007855628000049.jpg244151

Claims

1. A method for inducing modification of a target nucleic acid sequence in higher plant cells, To obtain artificial and / or non-naturally occurring type II Cas9 / RNA complexes by preparing a composition comprising recombinant Cas9 protein, guide RNA, and a Cas9 / RNA complex formed by at least a portion of the recombinant Cas9 protein and the guide RNA; and Introducing the aforementioned Cas9 / RNA complex into higher plant cells, Includes, The guide RNA, which includes the crRNA and tracrRNA portions, is either in vitro transcribed RNA or synthetic RNA. The target nucleic acid sequence, which is endogenous DNA, includes a portion complementary to the crRNA portion of the guide RNA. In the above composition, the guide RNA is in at least a 2-fold molar excess relative to the recombinant Cas9 protein. The Cas9 / RNA complex is a combination of the recombinant Cas9 protein and the guide RNA, and the Cas9 / RNA complex forms before being introduced into higher plant cells. The aforementioned method.

2. Guide RNA, (i) Dual guide RNA containing crRNA and tracrRNA; or (ii) Single-stranded guide RNA containing crRNA fused with tracrRNA The method according to claim 1.

3. A composition for use in a method of introducing a Cas9 / RNA complex into higher plant cells to induce modification of a target nucleic acid sequence in the higher plant cells, comprising a recombinant Cas9 protein, a guide RNA, and an artificial and / or non-spontaneously occurring type II Cas9 / RNA complex formed by at least a portion of the recombinant Cas9 protein and the guide RNA, wherein the complex is (a) Cas9 protein, and (b) Guide RNA containing the crRNA and tracrRNA portions It contains and forms a complex before being introduced into higher plant cells. The aforementioned guide RNA is RNA transcribed in vitro or synthetic RNA. The target nucleic acid sequence, which is endogenous DNA, includes a portion complementary to the crRNA portion of the guide RNA. The guide RNA is in at least a 2-fold molar excess relative to the recombinant Cas9 protein. The aforementioned composition.

4. Guide RNA, (i) Dual guide RNA containing crRNA and tracrRNA; or (ii) Single-stranded guide RNA containing crRNA fused with tracrRNA The composition according to claim 3.

5. The method according to any one of claims 1 to 2, wherein the target nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by Cas9, and the PAM is composed of a trinucleotide 5'-NGG-3'.

6. The composition according to any one of claims 3 to 4, wherein the target nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by Cas9, and the PAM is composed of a trinucleotide 5'-NGG-3'.

7. The method according to any one of claims 1 to 2, wherein the crRNA comprises two additional guanine nucleotides at its 5' end.

8. The composition according to any one of claims 3 to 4, wherein the crRNA comprises two additional guanine nucleotides at its 5' end.

9. The method according to any one of claims 1 to 2, wherein the Cas9 protein includes a nuclear localization signal (NLS), and the NLS is located at the N-terminus or C-terminus of the Cas9 protein.

10. The composition according to any one of claims 3 to 4, wherein the Cas9 protein contains a nuclear localization signal (NLS), and the NLS is located at the N-terminus or C-terminus of the Cas9 protein.

11. The method according to any one of claims 1 to 2, wherein the crRNA is 20 nucleotides long.

12. The composition according to any one of claims 3 to 4, wherein the crRNA is 20 nucleotides long.

13. The method according to any one of claims 1 to 2, wherein the modification comprises one of at least one nucleotide deletion, insertion, substitution, or indel.

14. The composition according to any one of claims 3 to 4, wherein the modification comprises one of at least one nucleotide deletion, insertion, substitution, or indel.

15. The method according to any one of claims 1 to 2, wherein the molar ratio of guide RNA to recombinant Cas9 protein is 29:14.0 to 29:1.

4.

16. The composition according to any one of claims 3 to 4, wherein the molar ratio of guide RNA to recombinant Cas9 protein is 29:14.0 to 29:1.4.

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