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

Abstract

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms 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.