对基因组进行定点敲除、插入和替换是研究基因功能和代谢工程的重要手段，但是传统的乳酸菌基因组修饰工具存在效率低、实验周期长、表型不稳定等缺陷。II型CRISPR/Cas系统是目前建立的一种新型基因组编辑工具，已经成功的应用于原核和真核生物的基因组靶向修饰。本项目拟利用这种系统的关键元件Cas9、sgRNA，构建重组表达质粒pGCs，建立乳酸乳球菌基因组靶向编辑平台；以绿色荧光蛋白为报告基因，对基因组打靶，通过检测荧光值及基因的转录水平，确定荧光蛋白的定点插入效率及最优打靶条件；在此基础上，利用源自枯草芽孢杆菌的血红素合成基因簇（hemeALBCD、hemEY）分步打靶基因组，分析菌株血红素合成能力与有氧条件下的生物量及细胞存活性，进一步证明II型CRISPR/Cas系统对乳球菌基因组定向编辑的有效性，建立新型的乳酸菌基因组靶向编辑技术，为乳酸菌的遗传改造和代谢途径优化提供高效准确的操作工具。

Site-directed deletion, insertion and replacement in genome are important strategies for exploring gene function and metabolic pathway, while the traditional genetic manipulation tools of lactic acid bacteria exhibit some limits, including low efficiency, long period and instable phenotype. The type II CRISPR/Cas system, a new emerging genome editing tool, has been successfully used in targeting-modification of prokaryotes and eukaryotes. In this project, two key elements, Cas9 and sgRNA, were used to develop a recombinant plasmid pGCs for construction of genome editing platform for Lactococcus lactis. With green fluorescent protein (gfp) gene as a reporter, the L. lactis genome was targeted. The efficiency of gene insertion and optimum targeting condition were investigated by fluorescence and transcriptional level. Then, heme biosynthetic gene clusters (hemeALBCD, hemEY) from Bacillus subtilis was used to target the genome. Through analysis of heme synthesis, biomass accumulation and cell survival under aerobic condition, the availability of type II CRISPR/Cas system for genetic modification in L. lactis was further verified. This study constructs a novel targeted genome editing technology, which could be applied for genetic modification and metabolic pathway optimization in lactic acid bacteria.