CRISPR-Cas9 allosteric inhibition by the viral anti-CRISPR protein AcrIIA6

International audienceIn the battle for survival between bacteriophages (phages) and their preys, phages have evolved anti-CRISPR proteins to neutralize the well-known bacterial CRISPR-Cas9 immune system. The striking sequence diversity of anti-CRISPR proteins and the lack of homology with proteins of known function raise questions relating to their modes of action. Moreover, anti-CRISPR proteins, as natural CRISPR-Cas9 inhibitors, hold great promise as biotechnological tools to fine-tune CRISPR-Cas9-based gene edition, and as useful addition to phage therapy. In this context, we embarked on the structural and functional characterization of the anti-CRISPR protein AcrIIA6, discovered in virulent phages infecting Streptococcus thermophilus, combining cryo-electron microscopy, in vitro analyses of macromolecular interactions, and functional assays in cells. We showed that AcrIIA6 uses a unique molecular mechanism to inhibit S. thermophilus Cas9 (St1Cas9). AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. It affects St1Cas9 conformational dynamics, which reduces St1Cas9 binding affinity for DNA and prevents St1Cas9 binding to its target within cells. Interestingly, these findings led us to identify a natural variant of St1Cas9 resistant to AcrIIA6, illustrating anti-CRISPR-driven mutational escape and molecular diversification of Cas9 proteins