State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, Shandong University, 27 Binhai Road, Qingdao, 266237, China.
Biotechnol Lett. 2021 Feb;43(2):495-502. doi: 10.1007/s10529-020-03024-7. Epub 2020 Oct 13.
To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei.
Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%.
Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.
构建方便的 CRISPR/Cas9 介导的基因组编辑系统，用于工业酶产生真菌草酸青霉和里氏木霉。
利用黑曲霉的 5S rRNA 启动子表达向导 RNA，使用带有 40bp 同源臂的供体 DNA 或不携带选择标记基因的供体 DNA 对草酸青霉中的β-葡萄糖苷酶基因 bgl2 进行缺失。使用无标记供体 DNA 作为编辑模板，在 creA 基因中实现了精确的小区域替换。在里氏木霉中，当用于基因编辑时，黑曲霉的 5S rRNA 启动子不如在草酸青霉中的效率高。使用天然的 5S rRNA 启动子，使用带有编辑效率为 36.67%的无标记供体 DNA，在 lae1 编码区引入了终止密码子。
通过使用异源或天然 5S rRNA 启动子表达向导 RNA，在丝状真菌草酸青霉和里氏木霉中开发了高效的基因组编辑系统。
