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 pint 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 .- 41 . (canceled) 
     
     
         42 . An engineered Cas9-crRNA complex comprising a Cas9 protein and at least one crRNA engineered to guide the Cas9-crRNA complex to a target double-stranded DNA molecule having a proto-spacer sequence and a proto-spacer adjacent (PAM) sequence, the Cas9-crRNA complex functioning to cleave the target sequence under suitable conditions. 
     
     
         43 . The complex of  claim 42  wherein the suitable conditions are in vitro. 
     
     
         44 . The complex of  claim 42  further comprising a tracrRNA. 
     
     
         45 . The complex of  claim 42 , wherein the crRNA comprises an approximately 20 nucleotide fragment complementary to a proto-spacer sequence in the target double-stranded DNA molecule. 
     
     
         46 . The Cas9-crRNA complex of  claim 42 , wherein the target DNA sequence is a plasmid DNA. 
     
     
         47 . The Cas9-crRNA complex of  claim 42 , wherein the target DNA sequence is a human genomic DNA. 
     
     
         48 . The Cas9-crRNA complex of  claim 42 , wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence. 
     
     
         49 . The Cas9-crRNA complex of  claim 48 , wherein the additional amino acid sequence is used for purification of the fusion polypeptide by affinity chromatography. 
     
     
         50 . The Cas9-crRNA complex of  claim 42 , wherein the Cas9 protein contains a point mutation in the RuvC active site motif. 
     
     
         51 . The Cas9-crRNA complex of  claim 50 , wherein the point mutation is D31A. 
     
     
         52 . The Cas9-crRNA complex of  claim 42 , wherein the Cas9 protein contains a point mutation in the HNH active site motif. 
     
     
         53 . The Cas9-crRNA complex of  claim 52 , wherein the point mutation is N891A.