To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

为了了解2类Cas蛋白的RuvC催化域如何切割DNA，有必要阐明处于催化作用状态的含RuvC的Cas复合物的结构。Cas12i2是2类VI型CRISPR-Cas内切核酸酶，可通过未知机制切割目标dsDNA。在这里，我们报告了Cas12i2–crRNA–DNA复合物和Cas12i2–crRNA复合物的结构。我们揭示了由Cas12i2识别和切割DNA的机制，以及由Helical-II域的构象变化诱导的RuvC催化口袋的活化。种子区域（核苷酸1–8）对于RuvC激活是必不可少的，但是需要中央间隔区的双链体（核苷酸9–15）。我们捕获了Cas12i2的催化状态，金属离子和ssDNA底物都结合在RuvC催化口袋中。一起，