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 .- 68 . (canceled) 
     
     
         69 . A method for site-specific modification of a target DNA molecule, the method comprising,
 contacting the target DNA molecule with an engineered Cas9-crRNA complex comprising a Cas9 protein, a crRNA, and a tracrRNA,   wherein the crRNA is engineered to guide the Cas9-CRISPR complex to a region comprising the site in the target DNA molecule.   
     
     
         70 . The method of  claim 69 , wherein the modification occurs either in vivo or in vitro. 
     
     
         71 . The method of  claim 70 , wherein the in vivo modification occurs in a mammalian cell. 
     
     
         72 . The method of  claim 69 , wherein a fragment of the crRNA is substantially complementary to the target DNA molecule. 
     
     
         73 . The method of  claim 72 , wherein the fragment of the crRNA that is substantially complementary to the target DNA molecule comprises about 20 nucleotides. 
     
     
         74 . The method of  claim 69 , wherein the Cas9 protein comprises at least one of an RuvC active site motif and an HNH active site motif. 
     
     
         75 . The method of  claim 69 , wherein the Cas9 protein has at least 80% identity with SEQ ID NO: 1. 
     
     
         76 . The method of  claim 69 , wherein crRNA comprises a 3′ and a 5′ region, wherein the 3′ region comprises at least 22 nucleotides of a CRISPR repeat and the 5′ region comprises at least 20 nucleotides of a spacer sequence engineered to be substantially complementary to a portion of the target DNA. 
     
     
         77 . The method of  claim 69 , wherein tracrRNA comprising a 5′ and 3′ region wherein at least a portion of the 5′ region is complementary to the 3′ region of crRNA. 
     
     
         78 . The method of  claim 69 , wherein the target DNA molecule comprises a proto-spacer adjacent motif (PAM) sequence upstream of a proto-spacer sequence. 
     
     
         79 . The method of  claim 78 , wherein the PAM sequence comprises a nucleic acid molecule having the nucleic acid sequence 5′-NGGNG. 
     
     
         80 . The method of  claim 69 , wherein the site-specific modification of the target DNA molecule is cleavage of the target DNA molecule. 
     
     
         81 . The method of  claim 74 , wherein the Cas9 protein contains a point mutation in the RuvC motif or the HNH motif, and wherein the modification of the target DNA molecule is site-specific nicking of the target DNA molecule. 
     
     
         82 . The method of  claim 81 , wherein the point mutation in the RuvC motif is D31A and the point mutation in the HNH motif is N891A. 
     
     
         83 . The method of  claim 69 , wherein the target DNA is double stranded or single stranded. 
     
     
         84 . The method of  claim 69 , wherein the Cas9 and/or crRNA is generated by recombinant DNA technology, in vitro translation or is chemically synthesized. 
     
     
         85 . The method of  claim 69 , wherein crRNA has a sequence comprising 5′-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUGUGUUGUUUCG-3′ (SEQ ID NO: 15) with any desirable spacer sequence. 
     
     
         86 . The method of  claim 69 , further comprising addition of RNase III polypeptide to the complex. 
     
     
         87 . A method for directing a Cas9-RNA-mediated double stranded cleavage of a target DNA molecule in a cell, the method comprising,
 contacting a target DNA molecule in a cell with a recombinant Cas9-crRNA complex comprising a Cas9 protein, an engineered crRNA, and a tracrRNA,   wherein the engineered crRNA is engineered to guide the Cas9-CRISPR complex to the target DNA molecule.   
     
     
         88 . The method of  claim 87 , wherein the engineered crRNA contains a 20 nucleotide fragment having substantial complementarity to the target DNA. 
     
     
         89 . The method of  claim 87 , wherein the cell is a mammalian cell. 
     
     
         90 . A method for directing a Cas9-RNA-mediated homologous recombination (HR) at a target DNA site in a cell, the method comprising,
 contacting a target DNA molecule in a cell with an engineered Cas9-crRNA complex comprising a Cas9 protein, a crRNA, and a tracrRNA, wherein the crRNA is engineered to guide the engineered Cas9-CRISPR complex to the target DNA molecule; and   a recombinant nucleic acid construct comprising a first and a second site of homology flanking the target site.   
     
     
         91 . The method of  claim 90 , wherein the crRNA is engineered to contain about 20 nucleotides having substantial complementarity to the target DNA. 
     
     
         92 . The method of  claim 90 , wherein the cell is a mammalian cell. 
     
     
         93 . The method of  claim 90 , wherein the target DNA contains a protospacer sequence that is least 80% complimentary to a spacer sequence in the crRNA in the complex, and a protospacer adjacent motif (PAM) sequence NGGNG downstream from the proto-spacer sequence, and wherein the Cas9 protein cleaves both target DNA strands at a cleavage site located 4 nucleotides upstream of the PAM sequence to create blunt ends. 
     
     
         94 . The method of  claim 90 , further comprising addition of RNase III polypeptide to the complex. 
     
     
         95 . A programmable Cas9-crRNA system comprising:
 a complex comprising a Cas9 protein,   a crRNA polynucleotide comprising a 3′ region and a 5′ region wherein the 3′ region comprises a repeat sequence present in a CRISPR locus and the 5′ region comprises at least 20 nucleotides of an engineered spacer sequence immediately downstream of the repeat in the CRISPR locus, and   a tracrRNA polynucleotide comprising a 5′ region and a 3′ region wherein at least a portion of the 5′ region is complementary to the 3′ region of the crRNA,   
       wherein the spacer sequence is engineered to direct the Cas9-CRISPR system to a target DNA molecule having a protospacer adjacent motif sequence. 
     
     
         96 . The programmable Cas9-crRNA system of  claim 95 , wherein the Cas9 protein comprises at least one of an RuvC active site motif and an HNH active site motif. 
     
     
         97 . The programmable Cas9-crRNA system of  claim 96 , wherein the Cas9 protein contains a point mutation in the RuvC motif and/or a point mutation in the HNH motif. 
     
     
         98 . The programmable Cas9-crRNA system of  claim 97 , wherein the point mutation in the RuvC motif is D31A and the point mutation in the HNH motif is N891A. 
     
     
         99 . The programmable Cas9-crRNA system of  claim 95 , wherein the 3′ region of the crRNA comprises at least 22 nucleotides of a repeat sequence present in a CRISPR locus. 
     
     
         100 . The programmable Cas9-crRNA system of  claim 95 , wherein the system is formed in vivo by introducing at least one plasmid encoding the Cas9 protein, the crRNA polynucleotide, and the tracrRNA polynucleotide into a microorganism, to result in a genetically modified microorganism, and isolating the complex from the genetically modified microorganism. 
     
     
         101 . The programmable Cas9-crRNA system of  claim 100 , further comprising incubating the isolated Cas9 protein, crRNA polynucleotide, and tracrRNA polynucleotide under conditions suitable for complex assembly. 
     
     
         102 . The programmable CAs9-crRNA system of  claim 100 , wherein the Cas9 protein, the crRNA polynucleotide, and the tracrRNA polynucleotide are encoded in two or three separate plasmids. 
     
     
         103 . The programmable Cas9-crRNA system of  claim 95 , wherein the system is formed in vitro by incubating the components of the system under conditions suitable for complex assembly. 
     
     
         104 . The programmable Cas9-crRNA system of  claim 103 , wherein the crRNA polynucleotide is obtained by in vitro transcription from a DNA fragment containing a single repeat-spacer-repeat unit, where the spacer has any desirable sequence, or is chemically synthesized. 
     
     
         105 . The programmable Cas9-crRNA system of  claim 95 , wherein the components comprise Cas9 polypeptide (SEQ ID NO: 1), tracrRNA polynucleotide (SEQ ID NO: 5), and crRNA polynucleotide (5′-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUGUGUUGUUUCG-3′) (SEQ ID NO: 15) with any desirable spacer sequence. 
     
     
         106 . The programmable Cas9-crRNA system of  claim 95 , further comprising addition of RNase III polypeptide to the complex of the system.