利用CRISPR/Cas系统建立新型乳酸菌基因组编辑技术

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.

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。

乳酸菌是重要的工业菌株，同时也是动物体内的正常菌群。对乳酸菌代谢改造和新功能的开发需要对其基因组进行编辑，然而传统操作体系存在效率低、突变表型不稳定的缺陷，亟待开发高效的遗传操作工具。因此本研究以CRISPR/Cas9系统在乳酸菌中的适用性为出发点，结合原噬菌体同源重组系统、Cre/loxP位点特异性重组系统开发了一系列可用于乳酸乳球菌和干酪乳杆菌基因组靶向突变、基因删除和插入的工具盒。.首先，将CRISPR/Cas9系统的关键元件在乳酸乳球菌和干酪乳杆菌中重组表达并打靶多个基因组位点，建立乳酸菌基因组打靶平台；结合原噬菌体重组酶RecT高效的在乳酸乳球菌NZ9000中完成目的基因的定点突变、小于100bp片段无痕删除和34bp loxP序列的插入。鉴定新型的同源重组系统LCABL_13040-50-60，结合Cre/loxP位点特异性重组工具完成干酪乳杆菌基因组的点突变、小片段删除及绿色荧光蛋白基因的插入。利用Cre/loxP位点特异性重组工具建立大片段DNA插入体系，完成血红素合成基因簇在乳球菌基因组上的靶向插入。基于苯丙氨酸tRNA合成酶α亚基的构建乳酸菌反向筛选系统，提高同源重组和无缝突变的效率。以上为乳酸菌的遗传改造提供高效准确的新型基因组编辑工具。