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的组成及其作用机制存在差异。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基因簇及Cas9对PAM的识别序列与现有报道不同。本项目首先分析并确定CRISPR/Cas9系统的结构组成（包括Cas基因簇、CRISPR array）及特点；然后通过序列分析结合RNA测序、Northern blot实验证实tracrRNA序列，通过构建PAM随机文库结合质粒干扰实验确定Cas9识别的PAM序列；最后基于体内、体外基因编辑实验探究CRISPR/Cas9系统的作用机制。该工作可以深入了解微生物中CRISPR/Cas9系统的多样性，发现新的CRISPR/Cas系统，并为获得更好的基因编辑工具奠定理论基础。

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) is an acquired immune system existing in archaea and bacteria with the long term of evolution processes. The composition and function mechanism of CRISPR/Cas9 are different in different microorganisms. In our previous study, a CRISPR/Cas9 locus was identified in the genome of Alicyclobacillus tengchongensis D-1, and its CRISPR composition, Cas gene clusters, and PAM sequence were different from now reported. To study the CRISPR/Cas9 system from A. tengchongensis D-1, we first analyze the composition of CRISPR/Cas9 system including Cas gene clusters and CRISPR array; Then, sequencing analysis combined with RNA sequencing and Northern blot were used to validate tracrRNA, and PAM randomized library construction combined with plasmid interference experiment were employed to confirm PAM sequence which recognized by Cas9; At last, in vivo and in vitro genome editing experiments were employed to explore the mechanism of CRISPR/Cas9. Combined with all the results above, we try to understand the diversity of CRISPR/Cas9 system in different microorganims, find new type of CRISPR/Cas system and provide theoretical foundation for the future development of new genome editing tools.