Fig. 1

Simultaneous inactivation of pyrG and kusA in A. niger D and D353. a Schematic diagram of simultaneous disrupted mutagenesis of both pyrG and kusA mediated by integrating the donor DNA with 40-bp micro-homology arms via CRISPR/Cas9 system. The donor DNAs of MHi-pyrG1-hph and MHi-kusA-hph were co-transformed with linear sgRNA constructs (sgRNA-pyrG1 and sgRNA-kusA) and Cas9 expression plasmid pCas9-hyh into the protoplasts of A. niger D and D353. Two DSBs were generated by the Cas9 under the guide of sgRNA, and then were repaired by HR with the integration of donor DNAs. b, c Diagnostic PCR analysis of the selected pyrG deficient transformants. The expected sizes of PCR products of the mutants were 3240-bp (pyrG-g-F/pyrG-g-R1) and 2702-bp (kusA-g-F/kusA-g-R), when the selection marker hph cassette were correctly inserted at the loci of pyrG or kusA. The expected sizes of PCR products of the hosts were 1345-bp (pyrG-g-F/pyrG-g-R1) and 804-bp (kusA-g-F/kusA-g-R), when the hph markers were not inserted at the loci of pyrG or kusA. For A. niger D353, the PCR products of some mutants using pyrG-g-F/pyrG-g-R1 were smaller than the expected size. After sequencing theses PCR products, it’s found that the smaller inserted fragments were the sgRNA-pyrG1 expression cassette