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.
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.).
