利用单分子荧光手段揭示CRISPR-Cas蛋白靶向和剪切机制

CRISPR-Cas系统是细菌和古生菌进化形成的抵御入侵噬菌体和质粒的适应性免疫系统。其中第二类的CRISPR-Cas系统，只需要一个多结构域的蛋白和一条单链RNA的介导就可以完成对特定核酸的识别和切割，因而在基因编辑和基因工程等领域有广泛的应用前景。Cas9作为Cas家族的明星分子，受到了广泛地关注，并进行了大量系统地研究。现在对Cas9在sgRNA介导下识别和剪切靶标DNA的工作机制有了一定的认知。但近期有新的证据表明，Cas9还有许多功能有待验证、发掘和认知。而其它第二类的CRISPR-Cas蛋白，如Cpf1和C2c2的功能研究仍然处在初期阶段，还有大量的探索工作有待完成。本项目将以Cas9、Cpf1和C2c2为主要研究对象，结合单分子荧光手段和生化实验从微秒到分钟的时间尺度上表征Cas蛋白在底物搜寻、识别和剪切过程中的动力学信息和构型变化，并阐释Cas蛋白功能的分子机制。

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated(Cas) system is a RNA-guided prokaryotic immune system evolved to target foreign genetic elements such as plasmid, phage DNAs and RNAs. Among them, class 2 CRISPR associated (Cas) proteins are especially attractive. Because they use a single large Cas protein to degrade foreign nucleic acids. The simplicity and specificity of programmable DNA/RNA recognition and cleavage by CRISPR-Cas proteins enable applications of precision genome editing and manipulation across many areas of biology...Enormous efforts have been made to reveal molecular mechanisms governing target searching and cleavage of Cas9. Several recent evidences presented new functions of Cas9, which suggested that furthermore studies on Cas9 are still demanded. On the other hand, research works on other class 2 CRISPR-Cas9 proteins, such as Cpf1 and C2c2, are still in their early stages...CRISPR-Cas proteins mediated DNA/RNA recognition and cleavage is a highly dynamic multi-step process, including target searching, recognition, and cleavage. Now, we are developing and applying single-molecule and ensemble biochemical assays to examine these processes in details. We mainly focus on class 2 Cas proteins, such as Cas9, Cpf1, and C2c2. Our aims are to elucidate molecular mechanisms governing rapid target searching and specificity of target recognition and cleavage and to provide guidance to improve and optimize CRISPR-Cas toobox.