RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX

Abstract

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule using an RNA-guided DNA endonuclease comprising at least one RNA sequence and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

1 - 78 . (canceled) 
     
     
         79 . A Cas9-crRNA complex comprising:
 a. a Cas9 protein;   b. a tracrRNA transcript; and   c. a CRISPR RNA transcript that is engineered to guide the Cas9-crRNA complex to a target DNA sequence for site specific modification of the target DNA sequence,   wherein (a), (b), and (c) are assembled in vitro.   
     
     
         80 - 113 . (canceled) 
     
     
         114 . The Cas9-crRNA complex of  claim 79 , wherein the crRNA comprises an approximately 20 nucleotide fragment complementary to a proto-spacer sequence in the target DNA sequence. 
     
     
         115 . The Cas9-crRNA complex of  claim 79 , wherein the target DNA sequence is a plasmid DNA. 
     
     
         116 . The Cas9-crRNA complex of  claim 79 , wherein the target DNA sequence is a human genomic DNA. 
     
     
         117 . The Cas9-crRNA complex of  claim 79 , wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence. 
     
     
         118 . The Cas9-crRNA complex of  claim 117 , wherein the additional amino acid sequence is used for purification of the fusion polypeptide by affinity chromatography. 
     
     
         119 . The Cas9-crRNA complex of  claim 79 , wherein the Cas9 protein contains a point mutation in the RuvC active site motif. 
     
     
         120 . The Cas9-crRNA complex of  claim 119 , wherein the point mutation is D31A. 
     
     
         121 . The Cas9-crRNA complex of  claim 79 , wherein the Cas9 protein contains a point mutation in the HNH active site motif. 
     
     
         122 . The Cas9-crRNA complex of  claim 121 , wherein the point mutation is N891A.