Gao Shuliang, Tong Yangyang, Wen Zhiqiang, Zhu Li, Ge Mei, Chen Daijie, Jiang Yu, Yang Sheng
School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai, 200234, China.
J Ind Microbiol Biotechnol. 2016 Aug;43(8):1085-93. doi: 10.1007/s10295-016-1789-8. Epub 2016 Jun 27.
Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.
解脂耶氏酵母被归类为一般认为安全（GRAS）的生物，是一种有大量文献记载的非常规酵母，已被广泛应用于多个工业领域以生产有价值的生化物质。本研究描述了一种用于解脂耶氏酵母基因组编辑的CRISPR-Cas9系统的构建，该系统使用单个质粒（pCAS1yl或pCAS2yl）将Cas9和相关的引导RNA表达盒（有或没有供体DNA）转运到靶基因。两个Cas9靶基因TRP1和PEX10通过非同源末端连接（NHEJ）或同源重组进行修复，在4天内，野生型菌株在解脂耶氏酵母中的最大效率为85.6%，ku70/ku80双缺陷菌株的最大效率为94.1%。通过NHEJ使用pCAS1yl实现了同时双基因和三基因编辑，效率分别为36.7%或19.3%，并且pCASyl系统成功扩展到不同的解脂耶氏酵母育种菌株。这种节省时间的方法将推动并改善解脂耶氏酵母的合成生物学、代谢工程和功能基因组学研究。
