Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Proc Natl Acad Sci U S A. 2020 Mar 24;117(12):6531-6539. doi: 10.1073/pnas.1917668117. Epub 2020 Mar 10.


Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.

Keywords: CRISPR; Cas9; anti-CRISPR; genome editing; self-targeting.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9 / antagonists & inhibitors*
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems* / genetics
  • Conserved Sequence
  • DNA / metabolism
  • Enzyme Inhibitors / metabolism*
  • Gene Editing
  • Genome, Bacterial / genetics
  • HEK293 Cells
  • Humans
  • Inverted Repeat Sequences
  • Staphylococcus / chemistry
  • Staphylococcus / genetics*
  • Staphylococcus aureus / enzymology


  • Bacterial Proteins
  • Enzyme Inhibitors
  • DNA
  • CRISPR-Associated Protein 9