There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

中文翻译：

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通过单分子反应通路分析定量评估工程化 Cas9 变体以增强靶标特异性

已经开发了许多工程化的 Cas9 变体，以最大限度地减少脱靶 DNA 的意外切割，但它们通过 DNA:RNA 异源双链化调节靶特异性的方式的详细机制仍然知之甚少。我们使用单分子 FRET 分析来跟踪 DNA 的动态：RNA 异源双链化的各种工程化 Cas9 变体与靶向和脱靶 DNA 相关。就像野生型 Cas9 一样，这些工程化的 Cas9 变体在其构象结构和核酸酶活性之间表现出很强的相关性。与野生型 Cas9 相比，随着碱基对错配的增加，可切割状态的比例下降得更快，这导致它们的靶标特异性增强。我们提出了一个反应模型来定量分析 R 环扩展连续过程中脱靶歧视的程度。我们发现关键的特异性增强步骤在 evoCas9 和 HypaCas9 的 DNA:RNA 异源双链化过程中被激活，而对于 Cas9-HF1、eCas9 和 Sniper-Cas9，它发生在异源双链化后阶段。这项研究揭示了 Cas9 靶向特异性背后的构象动力学，这将有助于加强其未来的合理设计原则。