2类CRISPR-Cas系统的校对机制
Proofreading mechanism of Class 2 CRISPR-Cas systems
摘要：
CRISPR系统会经历脱靶效应，干扰准确执行基因编辑的能力。虽然经验模型预测了特定平台中的脱靶效应，但基于目标识别基本过程的证据，CRISPR系统的广泛机制模型存在差距。在这项工作中，我们提出了一个第一性原理模型，在体内和体外进行了许多实验的支持。首先，我们使用截短的指导RNA在无细胞转录-翻译系统中观察和模拟SpCas 9-CRISPR靶识别过程的构象变化和离散的阶梯式DNA解旋事件，从而证实结构和FRET显微镜实验。其次，我们实现了一个能量景观来描述在spCas 9和Cas 12 a CRISPR系统中观察到的体内单错配效应。从错配分析结果中，我们发现CRISPR 2类系统通过利用间歇性能量屏障来维持其动力学校正机制，我们展示了如何利用景观来提高靶特异性。
英文摘要：
CRISPR systems experience off-target effects that interfere with the ability to accurately perform genetic edits. While empirical models predict off-target effects in specific platforms, there is a gap for a wide-ranging mechanistic model of CRISPR systems based on evidence from the essential processes of target recognition. In this work, we present a first principles model supported by many experiments performed in vivo and in vitro. First, we observe and model the conformational changes and discrete stepped DNA unwinding events of SpCas9-CRISPR target recognition process in a cell-free transcription-translation system using truncated guide RNAs, confirming structural and FRET microscopy experiments. Second, we implement an energy landscape to describe single mismatch effects observed in vivo for spCas9 and Cas12a CRISPR systems. From the mismatch analysis results we uncover that CRISPR class 2 systems maintain their kinetic proofreading mechanism by utilizing intermittent energetic barriers and we show how to leverage the landscape to improve target specificity.
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