CRISPR-Cas系统是原核生物所特有的获得性免疫系统，它在古菌和细菌中由向导RNA介导，来消灭外源核酸物质。对不同CRISPR-Cas系统分子机制的研究已经成为当前生物学研究领域的热点之一。近年来，申请人先后揭示了冰岛硫化叶菌III-B型CRISPR系统（Cmr-α）既有RNA干涉活性也有靶标RNA激活的DNA干涉活性（Peng et al., 2015, NAR; Han et al., 2017, NAR），但是其分子机理还有待深入研究。在本申请项目中，申请人将利用前期开发的基于内源CRISPR系统的基因组编辑技术（Li et al., 2016, NAR）构建冰岛硫化叶菌cmr基因突变株，并分析其Cmr-α系统RNP复合物的RNA和DNA切割活性。我们将解析Cmr-α复合物的组装及其识别靶标RNA的机制；并揭示由靶标RNA的结合促发RNP复合物构象变化进而激活DNA切割的机理。

The CRISPR-Cas system is the prokaryotic adaptive immunity that protects archaea and bacteria against invasion of viruses and plasmids. The system specifically eliminates nucleic acid invaders in a small guide RNA-dependent manner. Studying molecular mechanisms of different CRISPR-Cas systems has become one of the hottest research fields in contemporary biology. Research in the applicant group in the past few years has revealed that the type III CRISPR system (Cmr-α) has both RNA interference and RNA-activated DNA interference activity in Sulfolobus islandicus (Peng et al., 2015, Nucleic Acids Res; Han et al., 2017, Nucleic Acids Res), however, the molecular mechanisms involved in the process remain to be illustrated. In this project, we will employ the genetic and biochemical methods recently developed in our group to conduct the described research, i.e. constructing S. islandicus cmr gene mutants using the endogenous CRISPR-based genome editing (Li et al., 2016, Nucleic Acids Res) and using the RNA and DNA cleavage assay to analyze Cmr ribonucleoprotein (RNP) complexes purified from wild-type strain and cmr mutants. We aim to reveal the mechanism of RNP complex assembly, capture of target RNA and the conformation change of RNP complex to activate the DNA cleavage which activated by target RNA.