A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
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...> U6-St_tracrRNA(7-97) GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAA TTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATA ATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTA ACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTTACTT AAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGC AGGGTGTTTTCGTTATTTAA...
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...The Type II CRISPR system carries out targeted DNA double-strand break (DSB) in sequential steps (12-14, 20, 21)....
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Cites background from "A programmable dual-RNA-guided DNA ..."
...A recent in vitro reconstitution of the Streptococcus pyogenes type II CRISPR system demonstrated that crRNA fused to a normally trans-encoded tracrRNA is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA (4)....
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...S3) (4, 5), whereas inactivating both domains may enable Cas9 to function as a retargetable DNA binding protein....
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...Consistent with (4), in which a related Cas9 protein is shown to cut both strands 3 bp upstream of the PAM, our NHEJ data confirmed that most deletions or insertions occurred at the 3′ end of the target sequence (fig....
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...Existing studies of type II CRISPR specificity (4) suggest that target sites must perfectly match the PAM sequence NGG and the 8- to 12-base “seed sequence” at the 3′ end of the gRNA....
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...The ease of retargeting our system to modify genomic sequences greatly exceeds that of comparable ZFNs and TALENs, while offering similar or greater efficiencies (4)....
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References
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