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 DiTPR-CHAT_NTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

CRISPR-Cas7-11 是一种 III-E 型 CRISPR 相关核酸酶，可作为有效的 RNA 编辑工具。与 Cas/HEF1 相关信号转导器融合的四肽重复序列 (TPR-CHAT) 充当调节蛋白，与 CRISPR RNA (crRNA) 结合的 Cas7-11 相互作用，形成 CRISPR 引导的 caspase 复合物 (Craspase)。然而，通过TPR-CHAT精确调节Cas7-11的核酸酶活性以增强其效用还需要进一步研究。在这里，我们报告了Desulfonema ishimotonii ( Di ) Cas7-11-crRNA的冷冻电子显微镜 (cryo-EM) 结构，该结构与或不与 TPR-CHAT 全长或 N 末端复合。这些结构揭示了 Craspase 复合物的分子特征。结构分析结合体外核酸酶测定和电泳迁移率变动测定，揭示Di TPR-CHAT通过阻止靶RNA通过Di TPR-CHAT的N端65个氨基酸结合来负向调节Di Cas7-11的活性。 Di TPR-CHAT _NTD )。我们的工作表明，Di TPR-CHAT _{NTD可以作为}Di Cas7-11 调节器的一个小单元发挥作用，有可能实现安全应用，以防止 CRISPR-Cas7-11 系统的过度切割和脱靶效应。