新型生长快速需钠弧菌基因组无痕编辑体系构建

Construction of seamless genome editing system for fast-growing Vibrio natriegens

摘要:

需钠弧菌Vibrio natriegens作为近几年发展起来的一种新型生长快速底盘细胞，在合成生物学领域展现出良好的应用前景。基因组编辑是合成生物学研究中不可或缺的遗传操作手段。但是，开展需钠弧菌的合成生物学研究仍然有待进一步发展精准、高效的基因组编辑系统。针对这个问题，首先对6株需钠弧菌的生理表型进行检测，选取生长快速、表型稳定的CICC 10908菌株作为基因组编辑研究的宿主细胞。其次，建立并优化需钠弧菌自然转化系统。优化后的系统将筛选标记基因cat-sacB或KanR整合到需钠弧菌染色体上的同源重组效率分别达到4×10–5和4×10–4。再次，在优化的自然转化系统基础上，利用双向选择性筛选方法，建立了精准、高效的需钠弧菌基因组无痕编辑体系。通过测试，基因敲除、回补、插入和替换这4种不同类型基因编辑的阳性率分别为93.8%、100%、95.7%和100%。最后，需钠弧菌可以实现质粒的高效转化和消除。该工作为需钠弧菌合成生物学研究提供精准、高效的基因组无痕编辑手段。

Abstract:

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or KanR onto the chromosome of V. natriegens could reach 4×10–5 and 4×10–4, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.