Abstract

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse \"anti-CRISPR\" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional \"partner\" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an \"anti-anti-CRISPR.\" In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.