Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Nature. 2022 Sep;609(7925):191-196. doi: 10.1038/s41586-022-05114-0. Epub 2022 Aug 24.
Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy 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 and REC3 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 and REC3 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.
Cas9 是一种 CRISPR 相关的内切酶，能够进行 RNA 引导的、特异性的 DNA 切割。Cas9 的可编程活性已被广泛应用于基因组编辑应用中，但它对靶 DNA 结合和脱靶识别的精确机制仍不完全清楚。在这里，我们报告了一系列化脓性链球菌 Cas9 的冷冻电镜结构，这些结构捕获了靶向 DNA 杂交的定向过程。在 R 环形成的早期阶段，Cas9 的 REC2 和 REC3 结构域形成一个带正电荷的裂缝，容纳靶 DNA 双链的远端。引导物与靶物杂交超过种子区，导致 REC2 和 REC3 结构域的重排，并使 HNH 核酸酶结构域重新定位到催化无能力的检查点构象。在引导物-靶物异源双链体完成后，通过引导物-靶物异源双链体的扭曲，以及互补的 REC2 和 REC3 结构域的重排，使 HNH 核酸酶结构域的构象激活成为可能。这些结果共同为 Cas9 依赖于靶 DNA 的激活建立了一个结构框架，阐明了其构象检查点机制，并可能促进新型 Cas9 变体和具有增强特异性和活性的引导 RNA 设计的发展。
