利用单分子荧光手段揭示CRISPR-Cas蛋白靶向和剪切机制

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.

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。

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。本项目以第二类的CRISPR-Cas系统中的Cas9、Cas12a和CasX为主要研究对象，结合单分子荧光手段和生化实验动态表征了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，阐释了Cas蛋白功能的分子机制并指导改造和优化了Cas蛋白。..首先，揭示了Cas12a蛋白复合体切割双链DNA的动态调控机制，从而阐明了Cas12a具有比Cas9更高的切割特异性的分子机制。其次，发现了Cas9和Cas12a蛋白均利用三维和一维扩散实现高效快速地搜寻靶点，揭示了介导其一维扩散的非特异性结合位点，并展示了通过改造这些位点，可以提高Cas9和Cas12a的切割特异性。最后，通过对sgRNA 3’端的改造，实现了对Cas9结合DNA特异性的提高。..为了利用单分子荧光技术捕捉Cas蛋白在DNA上快速动态过程，本项目还发展了新型的scanning FRET-FCS，实现从纳秒到秒的检测时间窗口和亚纳米级的空间分辨率。还发展了基于非天然氨基酸和非天然碱基的蛋白质和核酸标记手段，扩展了单分子荧光技术的应用场景。