Inhibition of CRISPR-Cas9 with Bacteriophage Proteins

Benjamin J. Rauch, Melanie R. Silvis, Judd F. Hultquist, Christopher S. Waters, Michael J. McGregor, Nevan J. Krogan, Joseph Bondy-Denomy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

218 Scopus citations


Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific “anti-CRISPRs” present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

Original languageEnglish (US)
Pages (from-to)150-158.e10
Issue number1-2
StatePublished - Jan 12 2017


  • CRISPR-Cas
  • Cas9
  • Cas9 inhibitor
  • Listeria monocytogenes
  • anti-CRISPR
  • bacteriophage
  • dCas9
  • gene editing
  • prophage

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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