Fig. 3.

Overview of RNP‐assisted genome editing in filamentous fungi. Downscaling of the final optimized transformation procedure. Mature conidia were collected with 0.02% Tween 80. The germinated conidia are treated with 1 mg l⁻¹ benomyl on PDA plates to induce mitotic arrest (required for fungi with multinucleate protoplasts). After 15 h, mycelia were washed with sterilized water three times to remove benomyl. Protoplast preparation followed a previously reported procedure, and the protoplast concentration was diluted to 10⁶ ml^‐1. RNP(s) and donor DNA(s) (optional) were added to 200 μl protoplast suspensions. After addition of PEG 6000 solution, the surfactant Triton X‐100 is incorporated at a final concentration of 0.006% (w/v). The incubation time was prolonged to more than 25 min for full release of mitotic arrest (25 min for NHEJ, 50 min for HDR). Protoplast regeneration and transformant screening also followed a previously reported procedure. M: mitosis phase, G1: G1 phase in interphase, S: S phase in interphase, G2: G2 phase in interphase.