The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites via a single guide RNA (sgRNA). However, SpCas9 also binds and cleaves genomic off-target sites that are partially matched to the sgRNA. Here, we report a microscopic kinetic model that simultaneously captures binding and cleavage dynamics for SpCas9 and Sp-dCas9 in free-energy terms. This model not only outperforms state-of-the-art off-target prediction tools, but also details how Sp-Cas9's structure-function relation manifests itself in binding and cleavage dynamics. Based on the biophysical parameters we extract, our model predicts SpCas9's open, intermediate, and closed complex configurations and indicates that R-loop progression is tightly coupled with structural changes in the targeting complex. We show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our extensible approach can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

中文翻译：

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机械模型改善了脱靶的预测并揭示了SpCas9保真度的物理基础

化脓链球菌（Sp）Cas9核酸内切酶是重要的基因编辑工具。SpCas9通过单个向导RNA（sgRNA）定向到靶位点。但是，SpCas9还可以结合并切割与sgRNA部分匹配的基因组脱靶位点。在这里，我们报告了一个微观动力学模型，该模型同时捕获了以自由能表示的SpCas9和Sp-dCas9的结合和裂解动力学。该模型不仅优于最新的脱靶预测工具，而且详细介绍了Sp-Cas9的结构-功能关系如何在结合和裂解动力学中表现出来。基于我们提取的生物物理参数，我们的模型可以预测SpCas9的开环，中间和闭环复杂构型，并表明R环的进展与靶向复合物的结构变化紧密相关。我们显示，SpCas9靶向动力学针对扩展的序列特异性进行了调整，同时保持了靶向效率。我们的可扩展方法可以表征任何CRISPR-Cas核酸酶-基准针对SpCas9的自然和未来高保真变体；阐明CRISPR保真度的决定因素; 并揭示了提高工程系统特异性和效率的途径。