Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT  

作　　者：Tian Hong Qinghua Luo Haiyun Ma Xin Wang Xinqiong Li Chongrong Shen Jie Pang Yan Wang Yuejia Chen Changbin Zhang Zhaoming Su Haohao Dong Xiaodi Tang 

机构地区：[1]Department of Laboratory Medicine,State Key Laboratory of Biotherapy,National Clinical Research Center for Geriatrics,West China Hospital,Sichuan University,Chengdu,China [2]Frontiers Medical Center,Tianfu Jincheng Laboratory,West China Hospital,Sichuan University,Chengdu,China

出　　处：《Signal Transduction and Targeted Therapy》2024年第6期2716-2725,共10页信号转导与靶向治疗（英文）

基　　金：supported by the National Key Research and Development Program of China(2021YFA1301900,2021YFA1301203 and 2022YFC2303700 to H.D.and Z.S.);the National Natural Science Foundation of China(31900039 and 32170029 to X.T.,81971974 to H.D.,32222040 and 32070049 to Z.S.);the 1.3.5 Project for Disciplines Excellence of West China Hospital,Sichuan University(ZYYC20021 to H.D.);Tianjin Synthetic Biotechnology Innovation Capacity Improvement Action(TSBICIP-KJGG-008 to Z.S.).

摘　　要：CRISPR‒Cas7-11 is a Type Ⅲ-E CRISPR-associated nuclease that functions as a potent RNA editing tool.Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer(TPR-CHAT)acts as a regulatory protein that interacts with CRISPR RNA(crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex(Craspase).However,the precise modulation of Cas7-11’s nuclease activity by TPR-CHAT to enhance its utility requires further study.Here,we report cryo-electron microscopy(cryo-EM)structures of Desulfonema ishimotonii(Di)Cas7-11-crRNA,complexed with or without the full length or the N-terminus of TPR-CHAT.These structures unveil the molecular features of the Craspase complex.Structural analysis,combined with in vitro nuclease assay and electrophoretic mobility shift assay,reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT(DiTPR-CHAT_(NTD)).Our work demonstrates that DiTPRCHAT_(NTD) can function as a small unit of DiCas7-11 regulator,potentially enabling safe applications to prevent overcutting and offtarget effects of the CRISPR‒Cas7-11 system.

关 键 词：CRISPR crRNA TPR 

分 类 号：Q78[生物学—分子生物学]