Structural basis for the activity of the type VII CRISPR-Cas system

• 成果类型: Article
• 署名作者: Yang, Jie; Li, Xuzichao; He, Qiuqiu; Wang, Xiaoshen; Tang, Jingjing; Wang, Tongyao; Zhang, Yi; Yu, Feiyang; Zhang, Shuqin; Liu, Zhikun; Zhang, Lingling; Liao, Fumeng; Yin, Hang; Zhao, Haiyan; Deng, Zengqin; Zhang, Heng
• 署名单位: Tianjin Medical University; Chinese Academy of Sciences; Wuhan Institute of Virology, CAS; Chinese Academy of Sciences; University of Chinese Academy of Sciences, CAS; Wuhan University; Hubei Jiangxia Laboratory
• 刊物名称: Nature
• ISSN/ISSBN: 0028-6705
• DOI: 10.1038/s41586-024-07815-0
• 发表日期: 2024-09-12
• 关键词: metallo-beta-lactamase; r-loop formation; protein cleavage; cryo-em; craspase; complex

摘要

The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA1. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5 ' end, but not at the 3 ' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications. We describe the structure and activity of Cas14 nuclease, a component of the type VII CRISPR-Cas interference complex with Cas5 and Cas7, in different functional states.