Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage^1–3. The programmable activity of Cas9 has been widely utilized for genome editing applications^4–6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-EM structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2/3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2/3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

中文翻译：

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Cas9的R环形成和构象激活机制

Cas9 是一种 CRISPR 相关的核酸内切酶，能够进行 RNA 引导的位点特异性 DNA 切割^1-3。Cas9 的可编程活性已被广泛用于基因组编辑应用^4-6，但其靶 DNA 结合和脱靶辨别的精确机制仍未完全了解。在这里，我们报告了一系列化脓性链球菌的冷冻电镜结构Cas9 捕获目标 DNA 杂交的定向过程。在 R 环形成的早期阶段，Cas9 REC2 和 REC3 结构域形成一个带正电荷的裂隙，容纳目标 DNA 双链体的远端。通过种子区域的引导-靶标杂交诱导 REC2/3 结构域的重排和 HNH 核酸酶结构域的重新定位，以呈现无催化能力的检查点构象。指导目标异源双链的完成触发 HNH 核酸酶结构域的构象激活，这是通过指导目标异源双链的扭曲和互补 REC2/3 结构域重排实现的。一起，