Fig. 3

Titratable expression of pyrG mutagenesis constructed in A. niger. a Schematic diagram of pyrG titratalbe expression mutagenesis mediated by integrating the donor DNA with 40-bp micro-homology arms via CRISPR/Cas9 system based 5S rRNA. The donor DNA MHi-pyrG2-hyh:Tet-on, containing the Tet-on cassette, were co-transformed with linear sgRNA construct sgRNA-pyrG2 and Cas9 expression plasmid pCas9-hyh into the protoplasts of A. niger D353. DSBs at the locus of the upstream of pyrG encoding sequences, were generated by the Cas9 under the guide of sgRNA-pyrG2, and then were repaired by HR with the integration of donor DNA MHi-pyrG2-hyh:Tet-on, resulting in the replacement of pyrG native promoter. b Phenotypic screening of pyrG conditional expression mutants on solid plates. 1 × 10³, 1 × 10², and 1 × 10¹ spores were inoculated in 2 µL volumes onto the MM supplemented with various concentrations of Dox and MM with uridine as control. Plates were incubated at 30 °C in the dark for 48 h. Representative images are shown for technically triplicated experiments. Control, A. niger D353 as the positive controls; XMD1.6, the pyrG conditional expression mutants