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

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.