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 composition for inducing targeted disruption of endogenous genes in a eukaryotic cell, the composition comprising a Cas9/RNA complex comprising:
 a Cas9 protein linked to one or more tagging sequences, wherein the tagging sequence comprises a nuclear localization signal (NLS); and   a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: i) a portion to be hybridized with a portion of the tracrRNA, and ii) a portion complementary to a target DNA of the endogenous genes,   wherein the ribonucleoprotein functions as an endonuclease that induces targeted disruption of the target DNA.   
     
     
         59 . The composition of  claim 58 , wherein the Cas9 protein is complexed with the guide RNA to form a ribonucleoprotein before being introduced into the eukaryotic cell. 
     
     
         60 . The composition of  claim 58 , wherein the guide RNA is one of (a) a dual RNA and (b) a single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion. 
     
     
         61 . The composition of  claim 58 , wherein the Cas9 protein has the NLS in proximity to its N-terminal, its C-terminal, or both its N-terminal and C-terminal 
     
     
         62 . The composition of  claim 58 , wherein the Cas9 protein is derived from  S. pyogene.    
     
     
         63 . The composition of  claim 58 , wherein the tagging sequence further comprises a peptide tag. 
     
     
         64 . The composition of  claim 63 , wherein the peptide tag is a HA-tag. 
     
     
         65 . The composition of  claim 63 , wherein the peptide tag is a His-tag. 
     
     
         66 . The composition of  claim 58 , wherein the Cas9 protein is expressed in and purified from a bacterial system for a transgene-free use to avoid the host genome integration. 
     
     
         67 . The composition of  claim 58 , wherein the guide RNA is transcribed in vitro. 
     
     
         68 . The composition of  claim 58 , wherein the guide RNA is chemically synthesized. 
     
     
         69 . The composition of  claim 58 , wherein the target DNA comprises a first strand having a region complementary to the crRNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), wherein the PAM consists of the trinucleotide 5′-NGG-3′.