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

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

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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 SpyCas9 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 SpyCas9-sgRNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on SpyCas9.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 SpyCas9 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 SpyCas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of SpyCas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.

anti-CRISPRcryo-EMSpyCas9AcrⅡA25.1AcrⅡA32inhibiton

Jianlin Zheng、Yuwei Zhu、Tengjin Huang、Wenbo Gao、Jiale He、Zhiwei Huang

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HIT Center for Life Sciences,School of Life Science and Technology,Harbin Institute of Technology,Harbin 150080,China

Westlake Center for Genome Editing,Westlake Laboratory of Life Sciences and Biomedicine,School of Life Sciences,Westlake University,Hangzhou 310024,China

National Key Research and Development Program of ChinaNational Natural Science Foundation of ChinaTencent Foundation through the XPLORER PRIZENew Cornerstone Science FoundationHeilongjiang Touyan TeamNatural Science Foundation of Heilongjiang Province of China

2023YFF1000200U21A20276HITTY-20190034YQ2023C032

2024

10.1007/s11427-024-2607-8

中国科学:生命科学(英文版)
中国科学院

中国科学:生命科学(英文版)

CSTPCD
影响因子：0.806
ISSN：1674-7305
年,卷(期)：2024.67(9)