OBJECTIVE To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei. RESULTS 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%. CONCLUSIONS 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.

中文翻译：

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使用 5S rRNA 启动子驱动的引导 RNA 在草酸青霉和里氏木霉中进行 CRISPR/Cas9 介导的基因组编辑

目的在工业产酶真菌草酸青霉和里氏木霉中构建便捷的CRISPR/Cas9介导的基因组编辑系统。结果 使用来自黑曲霉的 5S rRNA 启动子进行引导 RNA 表达，使用携带 40-bp 同源臂的供体 DNA 或不包含选择标记基因的供体删除草酸假单胞菌中的 β-葡萄糖苷酶基因 bgl2。使用无标记供体DNA作为编辑模板，在creA基因中实现了小区域的精确替换。在里氏木霉中，当用于基因编辑时，黑曲霉 5S rRNA 启动子的效率低于草酸青霉。使用天然 5S rRNA 启动子，使用无标记供体 DNA 将终止密码子引入 lae1 编码区，编辑效率为 36.67%。