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 introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas complex, wherein the CRISPR/Cas complex comprises:
 a) a nucleic acid encoding a Cas9 polypeptide comprising a nuclear localization signal, wherein the nucleic acid is codon-optimized for expression in eukaryotic cells, and   b) a guide RNA that hybridizes to the target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans-activating crRNA (tracrRNA) portion,   
       whereby a site-specific, double stranded break at the target nucleic acid sequence is introduced. 
     
     
         59 . A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising contacting the target nucleic acid sequence with a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas complex, wherein the CRISPR/Cas complex comprises:
 a) a Cas9 polypeptide comprising a nuclear localization signal, and   b) a guide RNA that hybridizes to the target nucleic acid, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans-activating crRNA (tracrRNA) portion,   
       whereby a site-specific, double stranded break at the target nucleic acid sequence is introduced. 
     
     
         60 . The method of  claim 58  or  59 , wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide. 
     
     
         61 . The method of  claim 58  or  59 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         62 . The method of  claim 61 , wherein the mammalian cell is a human cell. 
     
     
         63 . The method of  claim 58  or  59 , wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell. 
     
     
         64 . The method of  claim 58 , wherein the nucleic acid encoding the Cas9 polypeptide is a vector. 
     
     
         65 . The method of  claim 58  or  59 , wherein the Cas9 polypeptide is a  Streptococcus  Cas9 polypeptide. 
     
     
         66 . The method of  claim 65 , wherein the Cas9 polypeptide is a  Streptococcus pyogenes  Cas9 polypeptide. 
     
     
         67 . The method of  claim 58  or  59 , wherein the nucleic acid encoding the Cas9 polypeptide is introduced into the eukaryotic cell before introducing the guide RNA into the eukaryotic cell. 
     
     
         68 . The method of  claim 58  or  59 , wherein the target DNA sequence comprises a first strand having a region complementary to the crRNA portion of the chimeric guide RNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), wherein the PAM consists of the trinucleotide 5′-NGG-3′.