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

作　　者：Jianlin Zheng Yuwei Zhu Tengjin Huang Wenbo Gao Jiale He Zhiwei Huang 

机构地区：[1]HIT Center for Life Sciences,School of Life Science and Technology,Harbin Institute of Technology,Harbin 150080,China [2]Westlake Center for Genome Editing,Westlake Laboratory of Life Sciences and Biomedicine,School of Life Sciences,Westlake University,Hangzhou 310024,China

出　　处：《Science China(Life Sciences)》2024年第9期1781-1791,共11页中国科学（生命科学英文版）

基　　金：supported by the National Key Research and Development Program of China(2023YFF1000200);the National Natural Science Foundation of China(U21A20276);the Tencent Foundation through the XPLORER PRIZE and the New Cornerstone Science Foundation to Z.H.,Heilongjiang Touyan Team(HITTY-20190034 to Z.H.);Natural Science Foundation of Heilongjiang Province of China(YQ2023C032 to Y.Z.)。

摘　　要：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.

关 键 词：anti-CRISPR CRYO-EM Spy Cas9 AcrⅡA25.1 AcrⅡA32 inhibiton 

分 类 号：Q939.4[生物学—微生物学]