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 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.
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Citations
53 Claims
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1-41. -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.
Specification