第二类CRISPR－Cas 系统中重要复合物的功能及活性调控

CRISPR-Cas系统是一种原核生物的获得性免疫系统，抵御外源核酸入侵。第二大类CRISPR-Cas系统通过Cas蛋白与crRNA结合形成复合物，该复合物特异性降解DNA/RNA，同时该复合物的活性收到多种小蛋白的调控。因此，第二大类CRISPR-Cas系统作为基因编辑工具被广泛应用。但是第二大类CRISPR-Cas系统的核心蛋白质-RNA 复合物降解目的核酸，以及受调控蛋白调控的分子机理还不明确；同时；CRISPR相关的RNA研究工具，以及控制基因编辑工具的调控开关还有待于开发。本项目以第二大类CRISPR-Cas系统的核心复合物为主要研究目标，采用X-射线晶体学和冷冻电镜技术，结合分子生物学、遗传学等方法开展核心蛋白复合物的结构与功能分析，阐明Csx等调控因子与anti-CRISPR蛋白的对第二大类CRISPR-Cas系统的激活或者抑制机理；优化CRISPR-Cas基因组编辑工具。

The CRISPR-Cas system is an adaptive immune system, protecting the bacterial and archae from the foreign nucleic acids. The key Cas protein assembles the guide RNA form the Cas-RNA complex, which recognizes the complementary target and degrades the target DNA or RNA. This activity of the Cas-RNA complex are regulated by the regulator of the host, and inhibited by anti-CRISPR proteins encoded by phages. To data, Cas9 protein in Class 2 CRISPR-Cas systems has been widely used as a genome-editing tool. Although previous studies have provided significant insights into the degradation of the invading DNA by the class 2 CRISPR-Cas systems, numerous questions are still elusive. In addition, all CRISPR genome-editing tools are developed based on their ability of dsDNA cleavage; no CRISPR- related RNA tools are available so far. Moreover, anti-CRISPR proteins that can act as the switch of genome-editing tool are required for further development of the CRISPR genome-editing tools. In this project, we will focus on the structural basis and molecular mechanism of class 2 CRISPR-Cas systems against invading nucleic acids. We will work on the structural and functional analysis of the core effector complexes by using X-ray crystallography and cryo-electron microscopy, together with molecular biology, genetics and other research methods. Importantly, we will also make efforts to gain insights in the regulatory factors of class 2 CRISPR-Cas system, to unravel the regulatory mechanism of Csx and anti-CRISPR proteins. Our results will facilitate the developing of the "switches" of gene editing, for better application of CRISPR-Cas system in genome editing and the treatment of diseases with the CRISPR tools.