超嗜热古菌火球菌Cas7蛋白的晶体结构与功能研究

Existing in the archaea and bacteria, the CRISPR-Cas system is a newly discovered foreign-DNA-defensing immunity system, which is composed of clustered regularly interspaced short palindromic repeats (CRISPR) and Cas proteins. CRISPR spacers contain the foreign DNA of bacteriophage and plasmids. Once the archaea or bacteria get invaded, the CRISPR locus will be transcribed into a long primary RNA, which will be further processed into a set of small CRISPR RNAs (crRNA). crRNA combines with Cas proteins into an Cascade (CRISPR-associated complex for antiviral defence) complex, which recognizes the target sequence in the invasive nucleic acid by base pairing. Consequently, the targeted sequence is degraded by Cas proteins in the complex. This research project focus on the studies of the.structure and function of the CRISPR-associated protein Cas7 of Pyrococcus furiosus (P.furiosus). Cas7 protein will be expressed using E. coli. The research process includes Cas7 extraction, purification and crystallization. The structure of Cas7 can be determined according to the diffraction data, collected at BL17U beamline of SSRF. According to the obtained structure of Cas7, the binding site for crRNA can be inferred. Experiment will be performed to understand the combination of Cas7 and crRNA, so that the functions of Cas7 in the Cascade.complex of the CRISPR-Cas system will be clear.

在古细菌和细菌中，由CRISPR和Cas蛋白组成的CRISPR-Cas系统是新近发现的一种抵御外源DNA入侵的免疫机制。CRISPR的间隔序列中含有源自噬菌体或者质粒的外源DNA序列，当细菌遭受新的入侵时，CRISPR会转录成一段长的前体RNA，前体RNA被加工成小的crRNAs，crRNAs与一系列Cas蛋白结合形成Cascade复合体，该复合体中的crRNAs通过碱基互补配对与靶标序列结合，最后由Cas蛋白将入侵的核酸降解。本课题以火球菌CRISPR-Cas系统中的Cas7蛋白为研究对象，在大肠杆菌中表达火球菌Cas7蛋白，提取纯化该蛋白后，生长蛋白晶体，在上海光源收集该蛋白晶体的衍射数据，解析得到该蛋白的晶体结构，基于晶体结构分析它与crRNA可能的结合位点，通过体外实验分析其与crRNA的结合特性，确定火球菌Cas7在形成CRISPR-Cas系统Cascade复合体中的重要作用。