Figure 3.

Efficient Mad7-induced mutagenesis in A. niger. (A) Scheme illustrating the two-phase transformant recovery scheme. Protoplasts transformed with a Mad7-CRISPR plasmid are mixed with melted medium containing low stringency hygromycin concentration and plated as the first layer; see Materials and Methods for details. When media solidifies, plates are incubated at 30 °C favoring growth of A. niger, or 37 °C favoring Mad7 activity. After one day of incubation, overlay medium containing high-stringency hygromycin concentration is added to form the second layer, and plates are then transferred for further incubation at the temperature indicated. (B) Mad7-induced mutagenesis of A. niger albA at a specific position indicated by a red X (left) and phenotypic consequence of mutation of colonies, which changes conidia color from wild-type black to mutant albA white (right). (C) Experiment showing Mad7-induced mutagenesis of albA. Transformation of protoplasts using the empty Mad7-CRISPR plasmid (indicated as control) or with the albA Mad7-CRISPR vector in three independent trials (indicated as T1–T3). All experiments were plated at two different temperatures as indicated. For trials T1–T3, numbers of transformants with specific phenotypes are indicated below plates: B, Black; H, Heterokaryon (black and white); W, White.