Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse “anti-CRISPR” proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1’s pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.

细菌CRISPR-Cas系统采用RNA引导的核酸酶破坏噬菌体（病毒）DNA。反过来，噬菌体已经进化出多种“抗CRISPR”蛋白（Acrs）来抵消获得性免疫。在单核细胞增生性李斯特菌中，原噬菌体编码两到三个不同的抗Cas9蛋白，而acrIIA1始终存在。但是，AcrIIA1的普及性及其机制的意义尚不清楚。在这里，我们报告AcrIIA1通过催化HNH结构域与Cas9高亲和力结合。李斯特菌属溶原发生期间，AcrIIA1触发Cas9降解。然而，在溶菌感染过程中，AcrIIA1由于其多步失活机制而无法阻断Cas9。因此，噬菌体编码一个快速结合并灭活Cas9的额外Acr。AcrIIA1还独特地抑制在李斯特菌（类似于SauCas9）和II-C型Cas9s中发现的高度差异的Cas9 ，这可能是由于Cas9 HNH域保守所致。总之，利斯特氏菌噬菌体使用可变的窄谱抑制剂在裂解生长中使Cas9失活，而广谱AcrIIA1刺激Cas9降解以保护溶原基因组。