CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide RNAs

• Published in: Biotechnology letters 2021-02, Vol.43 (2), p.495-502
• Main Authors: Wang, Qi, Zhao, Qinqin, Liu, Qin, He, Xin, Zhong, Yaohua, Qin, Yuqi, Gao, Liwei, Liu, Guodong, Qu, Yinbo
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Biochemistry; Biomedical and Life Sciences; Biotechnology; Cellobiase; Codons; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; Fungi; Gene Editing; Gene expression; Genetic modification; Genome editing; Genomes; Glucosidase; Homology; Hypocreales - genetics; Life Sciences; Microbiology; Original Research Paper; Penicillium - genetics; Penicillium oxalicum; Promoter Regions, Genetic - genetics; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Ribosomal, 5S - genetics; rRNA 5S; Trichoderma reesei; β-Glucosidase
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-020-03024-7

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.