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Annual Review of Biophysics

Volume 46, 2017

CRISPR–Cas9 Structures and Mechanisms

  • Fuguo Jiang1,2 and Jennifer A. Doudna1,2,3,4,5
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: [email protected], [email protected] 2California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720 3Department of Chemistry, University of California, Berkeley, California 94720 4Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 5Howard Hughes Medical Institute, University of California, Berkeley, California 94720
  • Vol. 46:505-529 (Volume publication date May 2017)
  • First published as a Review in Advance on March 30, 2017
  • © Annual Reviews

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 -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.

Keyword(s): Cas9, CRISPR, genome engineering, mechanism, off-target, structure
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2017-05-22
2025-06-19
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CRISPR–Cas9 Structures and Mechanisms
Annual Review of Biophysics 46, 505 (2017); https://doi.org/10.1146/annurev-biophys-062215-010822
/content/journals/10.1146/annurev-biophys-062215-010822
/content/journals/10.1146/annurev-biophys-062215-010822
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Supplementary Data

  • Supplemental Video 1: Conformational rearrangements of Cas9 upon sgRNA binding and target DNA recognition. This video shows a morph of the conformational changes Cas9 undergoes during guide RNA loading and DNA targeting in the following sequence: apo state (PDB ID 4CMP), sgRNA-bound pretargeting state (PDB ID 4ZT0), PAM-duplex DNA-bound state (PDB ID 4UN3), and dsDNA-bound state (PDB ID 5F9R). For clarity, the ssDNA-bound structure (PDB ID 4OO8) is not shown here, as it has a similar conformation to the PAM-duplex DNA-bound state.

    Supplemental Video 2: Structures of the Cas9--sgRNA bound to bona fide dsDNA substrates containing a canonical 5-TGG-3 PAM. This video highlights the path of the nontarget DNA strand in an active conformation with HNH nuclease domain positioned adjacent to the scissile phosphate within the target DNA strand. This is a conformation not observed in the ssDNA-bound and PAM duplex-bound structures and essential for concerted DNA cleavage.

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