Inhibition mechanisms of CRISPR-Cas9 by AcrⅡA25.1 and AcrⅡA32

In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.