Keywords:

Abstract:

<p>Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5 was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5 targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5 using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5 likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.</p>