CRISPR-Cascade在目标位点识别过程中的dsDNA解链机制

解析Cas蛋白与目标/脱靶位点的结合机制，将指导Cas蛋白低脱靶性的理性改造。Cas蛋白与目标/脱靶位点结合的一个前提是dsDNA解链。解链是个耗能过程，但Cas蛋白并非激酶，如何实现解链还不清楚。dsDNA解链是一瞬时过程，研究较为困难，该问题长期被CRISPR领域忽视，未得到解决。本项目将以Cascade为研究对象，解析其在位点识别过程中dsDNA的解链机制。根据项目负责人前期研究结果，推测PAM识别可诱导Cascade的构象变化，进而引起dsDNA的弯曲，导致了dsDNA的解链。本项目将利用单分子荧光能量共振转移技术对PAM识别、Cascade构象变化、DNA弯曲和解链之间的关系进行耦合性分析，解析Cascade在目标位点识别过程中dsDNA的解链机制。该研究将解决一个被CRISPR领域忽视的关键科学问题，为揭示其它Cas蛋白解链过程提供重要参考，指导Cas蛋白低脱靶性的理性改造。

Revealing Cas protein on-target/off-target binding mechanism will guide rational Cas protein engineering in reducing off-target effects. One of the prerequisites for Cas protein on-target/off-target binding is dsDNA unwinding. dsDNA unwinding requires energy, while Cas protein is not an ATPase/GTPase. The mechanism of how dsDNA is unwound by Cas protein without consuming ATP/GTP remains unclear. This problem has been ignored by the CRISPR filed because dsDNA unwinding during Cas protein target recognition process is transient and hard to analyze. This project will focus on dsDNA unwinding mechanism by Type I-E Cascade complex. Based on our previous results, we hypothesized that PAM recognition induces Cascade conformational change. Cascade conformational change will induce dsDNA bending and unwinding. In this project, we will use single-molecule FRET to directly visualize the relationship between PAM recognition, Cascade conformational change, DNA bending and unwinding. This project will reveal dsDNA unwinding mechanism by Cascade and provide a comprehensive kinetic model to understand dsDNA unwinding process by other Cas protein. Besides, this project will also provide information that can guide rational Cas protein engineering in reducing off-target effects and expanding PAM recognition.