Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6

Mol Cell. 2019 Dec 19;76(6):922-937.e7. doi: 10.1016/j.molcel.2019.09.012. Epub 2019 Oct 8.


In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Keywords: CRISPR-Cas9; St1Cas9; Streptococcus thermophilus Cas9; anti-CRISPR protein; bacteriophages; cryo-electron microscopy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Bacteriophages / genetics
  • Bacteriophages / metabolism*
  • Binding Sites
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Associated Protein 9 / ultrastructure
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / genetics
  • DNA / metabolism*
  • DNA / ultrastructure
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Humans
  • K562 Cells
  • Kinetics
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Streptococcus thermophilus / enzymology*
  • Streptococcus thermophilus / genetics
  • Structure-Activity Relationship
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Viral Proteins / ultrastructure


  • Viral Proteins
  • DNA
  • CRISPR-Associated Protein 9