crRNA介导的免疫系统的结构与功能研究

CRISPR/Cas是广泛存在于原核生物中的一种由crRNA介导干扰系统，分为I, II, III三种类型。在I型系统中，crRNA 结合5种Cas蛋白组成Cascade复合物，序列特异地识别外源入侵的DNA，然后招募Cas3蛋白并通过Cas3降解入侵核酸。在III型的III-A与III-B两个亚型中，cRNA 结合6种不同的Cas蛋白形成CSM或CMR复合物，抵御入侵的DNA或者RNA噬菌体。本项目通过研究I型和III型系统crRNA干扰过程中所形成的RNA-Cas蛋白复合物的结构和功能，重点包括I型系统中Cascade-DNA，Cascade-DNA-Cas3复合物的高分辨率结构，以及III型系统的CSM和CMR复合物结构，并结合ITC，质粒丢失等实验，分析CRISPR/Cas系统中功能复合物的组装方式与作用机理，从而为工程菌改造、超级细菌的治疗，以及作为基因组编辑工具提供依据。

CRISPR/Cas system is a crRNA-mediated interference pathway in the bacteria and archea. It can be classified into I, II, III three types. In the type-I system, crRNA binds five Cas proteins to form CRISPR-associated complex for antiviral defense (Cascade). Cascade complex will recognize target DNA and recruit degradation machine Cas3. Type-III can be further classified into III-A and III-B subtypes. crRNA binds 6 different Cas proteins to form CSM and CMR complex to defend the invading DNA and RNA viruses in subtypes III-A and III-B, respectively..In this project, we work on the structural and functional studies of RNA-protein complex in type I and III, combined with ITC and plasmid loss assay to provide the mechanism of crRNA interference. The major objects are the Cascade-DNA and Cascade-DNA-Cas3 complex in type-I, and CSM and CMR complex in type-III. The studies on the type-I and III will provide more insights into constructing engineering bacteria, killing super bacteria, and genome editing.