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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.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: jiangfg@berkeley.edu , doudna@berkeley.edu.
2
California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720.
3
Department of Chemistry, University of California, Berkeley, California 94720.
4
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
5
Howard Hughes Medical Institute, University of California, Berkeley, California 94720.

Abstract

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.

KEYWORDS:

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

[Indexed for MEDLINE]

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