论文标题
部分可观测时空混沌系统的无模型预测
Proofreading mechanism of Class 2 CRISPR-Cas systems
论文作者
论文摘要
CRISPR系统会经历脱靶效应，从而干扰了准确执行遗传编辑的能力。尽管经验模型可以预测特定平台中的脱靶效应，但基于目标识别基本过程的证据，广泛的CRISPR系统机械模型存在差距。在这项工作中，我们提出了由在体内和体外进行的许多实验支持的第一原理模型。首先，我们使用截短的指南RNA，确认结构和FRET显微镜实验，在无细胞的转录翻译系统中观察并模拟SPCAS9-CRISPR目标识别过程的构象变化和SPCAS9-CRISPR目标识别过程的离散DNA放松事件。其次，我们实施了一个能量景观，以描述SPCAS9和CAS12A CRISPR系统在体内观察到的单一不匹配效应。从不匹配分析结果中，我们发现CRISPR 2类系统通过利用间歇性的能量障碍来维护其动力学校对机制，我们展示了如何利用景观来提高目标特异性。
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.
