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 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.
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Citations
106 Claims
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1-68. -68. (canceled)
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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.
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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.
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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. - View Dependent Claims (91, 92, 93, 94)
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95. A programmable Cas9-crRNA system comprising:
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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, anda 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. - View Dependent Claims (96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106)
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Specification