Staphylococcus aureus Cas9 (SaCas9) is an RNA‐guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single‐molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6‐ and 18‐bp of PAM‐proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA‐bound SaCas9 a persistent block to DNA‐tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM‐distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.

中文翻译：

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金黄色葡萄球菌 Cas9 在 DNA 靶标结合和解离中的动态。


金黄色葡萄球菌Cas9 (SaCas9) 是一种 RNA 引导的核酸内切酶，以与原型间隔子相邻基序 (PAM) 相邻的互补 DNA 为目标进行切割。它的小尺寸有利于在各种生物体中进行基因组编辑的体内传递。在此，我们利用单分子和整体方法，系统地研究了 SaCas9 与 DNA 相互作用的机制。我们发现 SaCas9 的 DNA 结合和切割分别需要 6-bp 和 18-bp 的 PAM 近端 DNA 与引导 RNA 的互补性。这些活性是由三元复合物之间的两个稳定相互作用介导的，其中之一距离 PAM 约 6 bp，并且超出 SaCas9 在 DNA 上的明显足迹。值得注意的是，原型间隔子内的其他相互作用非常强，因此使 DNA 结合的 SaCas9 成为 DNA 跟踪马达的持久阻碍。有趣的是，切割后，SaCas9 自主释放 PAM 远端 DNA，同时保留与 PAM 的结合。这种部分 DNA 释放立即消除了其与原型间隔子 DNA 的强烈相互作用，从而促进其随后与 PAM 的解离。总的来说，这些数据提供了对 SaCas9 的动态理解并指导其有效应用。