CRISPR-Cas9构象激活与脱靶效应的分子模拟研究

细菌适应性免疫系统CRISPR-Cas9已经被开发为一种强大的靶向基因辑编工具。然而，该天然系统固有的脱靶效应构成其应用于临床治疗的最大瓶颈之一，并且它的编辑效率也有待进一步提高。在分子和原子尺度上，深入解析CRISPR-Cas9复合物的结构动力学性质与工作机理，无疑将有助于该技术的优化和改进。本项目运用常规分子动力学和增强分子动力学模拟并结合自由能计算和变构网络分析，深入研究CRISPR-Cas9的后期激活过程和脱靶效应的动学力性质与内在机制。研究的关键科学问题：一是弄清DNA底物与金属离子协同诱导Cas9催化构象形成的动力学机制和决定因素；二是从蛋白质变构的角度阐明CRISPR-Cas9靶向特异性和脱靶效应的分子机制。本研究将深化当前对CRISPR-Cas9复合物结构-动力学-功能关系的理解，为理性优化CRISPR-Cas9基因编辑技术以提高其靶向特异性和编辑活性提供重要的理论依据。

The bacterial immune system CRISPR-Cas9 has been developed as a powerful targeted gene-editing tool. However, the intrinsic off-target effect of this natural system constitutes a significant bottleneck for its therapeutic applications, and its editing efficiency also remains to be enhanced. At the molecular and atomic scale, deeply dissecting the structural and dynamical properties and working mechanism of CRISPR-Cas9 complex would undoubtedly benefit the optimization and improvement of this gene-editing technique. This project aims to provide an in-depth study on the dynamical characteristics and intrinsic mechanism underlying the CRISPR-Cas9 conformational activation at the later stage and off-target effect through conventional and enhanced molecular dynamics simulations in combination with free energy calculations and allosteric network analysis. Two key scientific questions are investigated: one is to clarify the dynamical mechanism and molecular determinants underlying the formation of Cas9 catalytic conformation that is cooperatively induced by DNA and metal ions; the other is to elucidate the molecular mechanism dictating the CRISPR-Cas9 targeting specificity and off-target effect from the perspective of protein allostery. This research would greatly deepen our current understanding of the structure-dynamics-function relationship of CRISPR-Cas9 complex and contribute important theoretical basis for rational optimization of the CRISPR-Cas9 gene-editing technique with improved targeting specificity and editing efficiency.