title: CRISPR-Cas9 Structures and Mechanisms.
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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: jiangfg@berkeley.edu , doudna@berkeley.edu.|California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720.
authors.forename: Fuguo
authors.initials: F
authors.lastname: Jiang
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: jiangfg@berkeley.edu , doudna@berkeley.edu.|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.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D064130:CRISPR-Associated Proteins; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA; D004247:DNA
keywords: CRISPR; Cas9; genome engineering; mechanism; off-target; structure