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 in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences 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 and using a cassette containing a single repeat-spacer-repeat unit.

What is claimed is: 
     
         1 - 49 . (canceled) 
     
     
         50 . A method for site-specific modification of a target DNA molecule, the method comprising:
 expressing a polynucleotide encoding a tracrRNA and an engineered crRNA to produce the tracrRNA and the engineered crRNA, wherein the engineered crRNA has a spacer sequence complementary to a nucleotide sequence of the target DNA molecule;   expressing a polynucleotide encoding a Cas9 protein heterologous to the tracrRNA and the engineered crRNA to produce the Cas9 protein; and   combining the tracrRNA and the engineered crRNA with the Cas9 protein to form a Cas9-crRNA complex;   wherein the Cas9-crRNA complex is reprogrammed to cleave the target DNA molecule, the Cas9-crRNA complex being heterologous to the target DNA molecule.   
     
     
         51 . The method of  claim 50 , further comprising preparing the polynucleotide encoding the tracrRNA and the engineered crRNA by chemical synthesis or in-vitro transcription. 
     
     
         52 . The method of  claim 50 , wherein the spacer sequence of the engineered crRNA comprises at least 20 nucleotides. 
     
     
         53 . The method of  claim 50 , wherein the Cas9 protein comprises a mutation in a RuvC active site motif or a HNH active site motif of the Cas9 protein. 
     
     
         54 . The method of  claim 50 , wherein the polynucleotide encoding the tracrRNA and the engineered crRNA is a plasmid DNA. 
     
     
         55 . The method of  claim 50 , further comprising contacting the Cas9-crRNA complex with the target DNA molecule to cleave the target DNA molecule. 
     
     
         56 . The method of  claim 55 , wherein the target DNA molecule is double stranded. 
     
     
         57 . A method for site-specific modification of a target DNA molecule, the method comprising:
 expressing a polynucleotide encoding an engineered crRNA to produce the engineered crRNA, wherein the engineered crRNA has a spacer sequence complementary to a nucleotide sequence of the target DNA molecule;   expressing a polynucleotide encoding a Cas9 protein that is heterologous to the engineered crRNA to produce the Cas9 protein; and   combining the engineered crRNA with the Cas9 protein and a tracrRNA to form a Cas9-crRNA complex, wherein the Cas9-crRNA complex is reprogrammed to cleave the target DNA molecule.   
     
     
         58 . The method of  claim 57 , further comprising contacting the Cas9-crRNA complex with the target DNA molecule. 
     
     
         59 . The method of  claim 58 , wherein the target DNA molecule is double stranded, and wherein the Cas9-crRNA complex modifies the target DNA molecule by site-specific double stranded cleavage of the target DNA molecule. 
     
     
         60 . The method of  claim 58 , wherein the target DNA molecule includes a protospacer-adjacent motif. 
     
     
         61 . The method of  claim 57 , wherein the Cas9 protein is a nickase. 
     
     
         62 . The method of  claim 57 , wherein the Cas9-crRNA complex is formed in the absence of RNaseIII. 
     
     
         63 . A method for site-specific modification of a target DNA molecule, the method comprising:
 re-programming a sequence of a polynucleotide to encode an engineered crRNA capable of site-specifically binding to a nucleotide sequence of the target DNA molecule;   expressing the polynucleotide to produce the engineered crRNA; and   combining the engineered crRNA with a tracrRNA, and a Cas9 protein heterologous to the engineered crRNA to form a Cas9-crRNA complex, wherein the Cas9-crRNA complex has engineered specificity towards the target DNA molecule.   
     
     
         64 . The method of  claim 63 , further comprising contacting the Cas9-crRNA complex with the target DNA molecule to cleave the target DNA molecule, wherein the Cas9-crRNA complex modifies the target DNA molecule by site-specific cleavage of the target DNA molecule. 
     
     
         65 . The method of  claim 64 , wherein the target DNA molecule is double stranded and includes a protospacer-adjacent motif. 
     
     
         66 . The method of  claim 63 , wherein the spacer sequence of the engineered crRNA comprises at least 20 nucleotides. 
     
     
         67 . The method of  claim 63 , wherein the polynucleotide encoding the engineered crRNA is prepared by chemical synthesis or in-vitro transcription. 
     
     
         68 . The method of  claim 63 , wherein the Cas9-crRNA complex is formed in the absence of RNaseIII.