Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA–DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.

Cas12i 是最近发现的 V 型 CRISPR-Cas 核酸内切酶，主要切割双链 DNA 底物的非目标链。Cas12i 的这种切口活动可能用于具有高特异性的基因组编辑。为了阐明其靶标识别和切割机制，我们确定了 Cas12i 在多种功能状态下的冷冻电镜结构。Cas12i 预先订购 crRNA 的七核苷酸种子序列用于目标识别，并通过 crRNA-DNA 杂交进行两步激活。14 个碱基对的形成激活了切口酶活性，28 bp 杂交促进了靶链的切割。获得的原子结构和机制见解应该有助于在基因组编辑应用中操纵 Cas12i。