COMPOSITION FOR CLEAVING A TARGET DNA COMPRISING A GUIDE RNA SPECIFIC FOR THE TARGET DNA AND CAS PROTEIN-ENCODING NUCLEIC ACID OR CAS PROTEIN, AND USE THEREOF

1 - 57 . (canceled)

58 . A method of modifying a target endogenous nucleic acid sequence, wherein the target endogenous nucleic acid sequence is in a nucleus of a eukaryotic cell, comprising:

introducing the Cas9/RNA complex into the eukaryotic cell, wherein the Cas9/RNA complex comprises a recombinant Cas9 protein and a guide RNA,

wherein the Cas9/RNA complex is a combination of the recombinant Cas9 protein and a guide RNA, wherein the guide RNA includes a crRNA and a tracrRNA,

wherein the guide RNA is transcribed in vitro or synthesized chemically,

wherein the target endogenous nucleic acid sequence includes a portion complementary to the crRNA of the guide RNA, and

wherein the combination of the recombinant Cas9 and the guide RNA produces a modification of the target endogenous nucleic acid sequence in the nucleus of the eukaryotic cell.

59 . The method of claim 58 , wherein the Cas9/RNA complex is introduced to the eukaryotic cell by transfection, wherein the transfection is performed by the method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, and polyethylene glycol (PEG)-mediated transfection of protoplasts.

60 . The method of claim 58 , wherein the Cas9/RNA complex is introduced to the nucleus of the eukaryotic cell by the transfection.

61 . The method of claim 58 , wherein the guide RNA is (i) a dual guide RNA comprising a crRNA and a tracrRNA; or (ii) a single-chain guide RNA comprising a crRNA fused to a tracrRNA.

62 . The method of claim 58 , wherein the target endogenous nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by the recombinant Cas9 protein, wherein the PAM consists of trinucleotide 5′-NGG-3′.

63 . The method of claim 58 , wherein the recombinant Cas9 protein comprises a nuclear localization signal (NLS), wherein the NLS is at N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the recombinant Cas9 protein.

64 . The method of claim 58 , wherein the crRNA is 20 nucleotides in length.

65 . The method of claim 58 , wherein the modification includes any one of deletion, insertion, substitution or indel of at least one of nucleotide.

66 . The method of claim 58 , further comprising inducing divisions of the eukaryotic cell to be divided into a plurality of cells which include the modified nucleic acid sequence.

67 . The method of claim 58 , wherein the combination of the recombinant Cas9 protein and the guide RNA is assembled by mixing and incubating the recombinant Cas9 protein and the guide RNA in vitro.

68 . The method of claim 58 , wherein the guide RNA is transcribed in vitro or synthesized chemically.

69 . The method of claim 58 , wherein the recombinant Cas9 protein is purified from a bacterial system for a plasmid-free used to avoid host genome integration.