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Annu Rev Biophys. 2017 May 22;46:505-529. doi: 10.1146/annurev-biophys-062215-010822. Epub 2017 Mar 30.

CRISPR-Cas9 Structures and Mechanisms.

Jiang F1,2, Doudna JA1,2,3,4,5.

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Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: ,
California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720.
Department of Chemistry, University of California, Berkeley, California 94720.
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Howard Hughes Medical Institute, University of California, Berkeley, California 94720.


Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.


CRISPR; Cas9; genome engineering; mechanism; off-target; structure

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