Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4_Hpa

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4_Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4_Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.