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 . A composition for cleaving target DNA in eukaryotic cells or organisms comprising a guide RNA specific for target DNA or DNA that encodes the guide RNA, and Cas protein-encoding nucleic acid or Cas protein. 
     
     
         2 . The composition of  claim 1 , wherein the target DNA is an endogenous target DNA. 
     
     
         3 . The composition of  claim 1 , wherein the guide RNA is a dualRNA comprising a crRNA and a tracrRNA. 
     
     
         4 . The composition of  claim 1 , wherein the guide RNA is a single-chain guide RNA (sgRNA). 
     
     
         5 . The composition of  claim 4 , wherein the single-chain guide RNA comprises portion of a crRNA and a tracrRNA. 
     
     
         6 . The composition of  claim 1 , wherein the guide RNA further comprises one or more additional nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA. 
     
     
         7 . The composition of  claim 1 , wherein the guide RNA further comprises 2 additional guanine nucleotides at the 5′ end of the single-chain guide RNA or the crRNA of the dualRNA. 
     
     
         8 . The composition of  claim 1 , which induces targeted mutagenesis in eukaryotic cells or organisms. 
     
     
         9 . The composition of  claim 1 , for use in the genotyping of a genome in the eukaryotic cells or organisms in vitro. 
     
     
         10 . The composition of  claim 1 , wherein the guide RNA and the Cas protein function as a pair, and wherein the pair comprises two guide RNAs which induce two nicks on different strands. 
     
     
         11 . The composition of  claim 1 , wherein the guide RNA is in the form of an isolated RNA, or is encoded in a vector, wherein the vector is a viral vector, plasmid vector, or  agrobacterium  vector. 
     
     
         12 . The composition of  claim 1 , comprising a guide RNA specific for the target DNA and Cas9 protein. 
     
     
         13 . The composition of  claim 1 , for cleaving the target DNA in eukaryotic cells or organisms ex vivo or in vivo. 
     
     
         14 . The composition of  claim 1 , wherein the Cas protein-encoding nucleic acid or the Cas protein is derived from the genus  Streptococcus.    
     
     
         15 . The composition of  claim 14 , wherein the genus  Streptococcus  is  Streptococcus pyogenes.    
     
     
         16 . The composition of  claim 15 , wherein the Cas protein recognizes the NGG trinucleotide. 
     
     
         17 . The composition of  claim 1 , wherein the Cas protein is Cas9 protein or variant thereof. 
     
     
         18 . The composition of  claim 1 , wherein the Cas protein is linked to a protein transduction domain. 
     
     
         19 . The composition of  claim 17 , wherein the variant of Cas9 protein is a mutant form of Cas9 in which the catalytic aspartate residue is changed to any other amino acid. 
     
     
         20 . The composition of  claim 19 , wherein the amino acid is an alanine. 
     
     
         21 - 57 . (canceled)