RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
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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.
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Citations
60 Claims
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1-41. -41. (canceled)
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42. A method for site-specific modification of a target DNA molecule, the method comprising
contacting a target DNA molecule with a recombinant Cas9-crRNA complex comprising a Cas9 protein, a crRNA, and a tracrRNA, wherein the crRNA guides the recombinant Cas9-CRISPR complex to a region comprising the site in the target DNA molecule.
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54. A programmable Cas9-crRNA system comprising:
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a recombinant nucleic acid molecule comprising a first polynucleotide encoding a Cas9 protein, a second polynucleotide encoding a crRNA 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, anda third polynucleotide encoding tracrRNA 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. - View Dependent Claims (55, 56, 57, 58, 59, 60)
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Specification