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

Jie Yang, Xuzichao Li, Qiuqiu He, Xiaoshen Wang, Jingjing Tang, Tongyao Wang, Yi Zhang, Feiyang Yu, Shuqin Zhang, Zhikun Liu, Lingling Zhang, Fumeng Liao, Hang Yin, Haiyan Zhao, Zengqin Deng () and Heng Zhang ()
Additional contact information
Jie Yang: Tianjin Medical University
Xuzichao Li: Tianjin Medical University
Qiuqiu He: Tianjin Medical University
Xiaoshen Wang: Tianjin Medical University
Jingjing Tang: Chinese Academy of Sciences
Tongyao Wang: Tianjin Medical University
Yi Zhang: Tianjin Medical University
Feiyang Yu: Wuhan University
Shuqin Zhang: Tianjin Medical University
Zhikun Liu: Tianjin Medical University
Lingling Zhang: Tianjin Medical University
Fumeng Liao: Tianjin Medical University
Hang Yin: Tianjin Medical University
Haiyan Zhao: Wuhan University
Zengqin Deng: Chinese Academy of Sciences
Heng Zhang: Tianjin Medical University

Nature, 2024, vol. 633, issue 8029, 465-472

Abstract: Abstract 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.

Date: 2024
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DOI: 10.1038/s41586-024-07815-0

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