原核生物CRISPR-Cas系统作为一种可遗传的适应性免疫系统可特异识别并清除外源病毒或质粒。但目前人们对于该系统在适应过程中如何特异地从病毒获取DNA片段并整合到CRISPR结构中形成新的spacer的复杂机制还很不清楚。最近，本实验室在嗜盐古菌CRISPR系统对病毒的适应过程中发现的“PAM验证的引发适应机制”很好地回答了适应过程的异己区分问题，但引发适应过程的分子细节及其特异性的分子机制还有待进一步阐明。本项目以嗜盐古菌I-B型CRISPR高效引发适应系统为研究对象，聚焦于其“靶向引发”“双向寻取”和“定点整合”三个关键阶段，结合分子遗传学技术、高通量测序和体外单分子观测分析等现代分子生物学手段，深入剖析I-B型CRISPR-Cas系统的引发适应过程，重点研究其关键步骤的特异性机制的分子基础，为深刻认识CRISPR-Cas系统的适应策略及发展CRISPR-Cas相关技术提供理论依据。

Prokaryotic adaptive immunity relies on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins to detect and destroy foreign nucleic acids (e.g. virus or plasmid). However, it is not clear how DNA sequences, termed protospacers, are discriminatively acquired from the virus and stored as new spacers into the CRISPR locus to guide the subsequent target interference. Recently, we showed that the primed adaptation, which hitches and cooperates with the interference pathway, could perfectly achieve the self vs. non-self discrimination through the priming-crRNA guidance and PAM recognition, whereas the molecular details remained to be elucidated. This project will focus on the molecular mechanisms for priming adaptation of the type I-B CRISPR-Cas system in haloarchaea. Combining genetic manipulation of the CRISPR DNA and Cas proteins, high-throughput analysis of new spacers and their PAMs, and single-molecule assays of the dynamic behaviors of related Cas proteins, we aim to dissect the I-B type priming adaptation process on the three key steps, i.e. the priming (to the target sequence), the bi-directional searching (for the new protospacers), and the site-specific spacer integration (into the CRISPR locus). We will focus on revealing the essential mechanistic specificities and related molecular determinants in each step, hoping to achieve deep insights into the adaptation strategy of CRISPR system, and to provide theoretical foundations for its application in phage control or in developing novel CRISPR-Cas related biotechnologies.