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.

新鉴定的VII型CRISPR-Cas候选系统使用由Cas5和Cas7蛋白形成的CRISPR RNA引导的核糖核蛋白复合物来靶向RNA ¹ 。然而，RNA 切割是由专用的 Cas14 核酸酶执行的，该酶与其他 CRISPR-Cas 系统的效应核酸酶不同。在这里，我们报告了 Cas14 结合干扰复合物在不同功能状态下的七个冷冻电子显微镜结构。 Cas14 是一种溶液中的四聚体蛋白，以靶标 RNA 依赖性方式被招募到 Cas5-Cas7 复合物中。 Cas14 的 N 端催化结构域结合一段底物 RNA 进行切割，而 C 端结构域主要负责将 Cas14 束缚到 Cas5-Cas7 复合物。生化裂解测定证实了捕获的功能构象，揭示了 Cas14 与 Cas5-Cas7 复合物上的不同位点结合以执行单独的裂解事件。值得注意的是，插入的 Cas7 精氨酸被 C 端结构域的 C 形夹夹在中间，精确调节 Cas14 结合。更有趣的是，靶标 RNA 的切割会被 5' 端的互补原型间隔区侧翼序列改变，但 3' 端则不会。总而言之，我们的研究阐明了 VII 型 CRISPR-Cas 系统中干扰复合物组装和底物裂解的关键分子细节，这可能有助于为生物技术应用合理设计 VII 型 CRISPR-Cas 系统。