CRISPR-Cas9 Structures and Mechanisms

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


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.

Publication types

  • Review

MeSH terms

  • CRISPR-Associated Proteins
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / chemistry*
  • DNA / genetics
  • RNA / chemistry*
  • RNA / genetics
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / genetics


  • CRISPR-Associated Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • RNA
  • DNA