Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing. [Display omitted] •Crystal structure of S. pyogenes Cas9 in complex with sgRNA and suppressor AcrIIA4•Selective recognition of pre-target bound SpyCas9 binary complex by AcrIIA4•Competitive binding of AcrIIA4 over dsDNA for SpyCas9-sgRNA binary complex•Mechanistic insights into blockage of SpyCas9 preventing dsDNA cleavage by AcrIIA4 Anti-CRISPRs are phage factors that inhibit bacterial CRISPR systems. Yang and Patel describe the structure of an anti-CRISPR protein, AcrIIA4, in complex with Cas9 with single-guide RNA. The structure reveals the mechanism of inhibition and provides the basis for developing tools to regulate Cas9 function in genome editing.