CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.

•The CRISPR/Cas9 system has been established in Penicillium subrubescens.•Efficient protoplast generation and transformation protocols have been implemented.•CRISPR/Cas9 enabled the deletion of ku70 gene in P. subrubescens.•ku70 mutation does not affect P. subrubescens growth at optimal temperature.•Δku70 strains showed similar protein production pattern to the wild type.