Fungal CRISPR-Cas9 Protocol

Escherichia coli strain DH5α was used to propagate all plasmids. The Aspergillus species used for implementation of CRISPR-Cas9 are listed in Table 2. Genomic DNA (gDNA) from fungal strains was isolated via FastDNA SPIN Kit for Soil DNA extraction kit (MP Biomedicals, USA). The mutant strains made in this study are listed in Table 1. All aspergilli were cultivated on standard solid glucose based minimal medium (MM) (1% glucose, 1x nitrate salt solution [41 ], 0.001% Thiamine, 1x trace metal solution [42 (link)], 2% agar), supplemented with 10 mM uridine (Uri), 10 mM uracil (Ura), and/or 4 mM L-arginine (Arg) when required. Solid plates containing 5-fluoroorotic acid (5-FOA) were made as MM+Arg+Uri+Ura supplemented with filter-sterilized 5-FOA (Sigma-Aldrich) to a final concentration of 1.3 mg/ml. For transformation media (TM) glucose was replaced with 1 M sucrose.