腾冲脂环酸芽孢杆菌D-1中AtCas9的结构特征及作用机制研究

CRISPR/Cas系统是细菌和古菌在长期进化过程中形成的一种获得性免疫防御机制。不同微生物中CRISPR/Cas9系统的组成及其作用机制存在差异，然而差异性产生的原因及其功能特异性有待于深入研究。申请人在前期研究中发现腾冲脂环酸芽孢杆菌D-1基因组中存在CRISPR/Cas9系统，其CRISPR组成、Cas基因簇、Cas9对PAM的识别序列以及对靶标DNA的切割方式与现有报道显著不同。本项研究首先解析AtCas9与向导RNA及靶标DNA三元复合物的蛋白结构；然后基于结构解析AtCas9识别PAM序列、向导RNA及靶标DNA的分子机制、关键结构域及氨基酸位点；最后基于体内、体外基因编辑实验探究AtCas9的作用机制。本研究为深入认识细菌CRISPR/Cas系统的多样性及其独特的抵抗异源核酸入侵机制具有重要科学意义，也为开发全新的基因编辑工具提供理论基础。

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system is an acquired immune defense mechanism formed in the long-term evolution of bacteria and archaea. The composition and function mechanism of CRISPR/Cas9 is different in different microorganisms. However, the reasons and mechanisms of these differences are need to be further investigated. In our previous study, a new CRISPR/Cas9 system from Alicyclobacillus tengchongensis D-1 was identified, and its CRISPR composition, Cas gene clusters, PAM recognition sequence and cleavage mechanism towards target DNA were different from now reported. To study this CRISPR/Cas9 system, we will first determine the structure of AtCas9 in complex with sgRNA and target DNA; Then, to resolve the recognition mechanism of AtCas9 to PAM, sgRNA and target DNA, and the critical domains and amino acids involved based on the AtCas9 structure; At last, to explore the mechanism of AtCas9 used in vivo and in vitro genome editing experiments. Combined with all the results above, it is of great scientific significance to further understand the diversity of CRISPR/Cas system and molecular mechanism of how bacteria resist heterologous nucleic acids invasion through CRISPR/Cas system. Meanwhile, it can also provide theoretical basis for the development of new genome editing tools.