Figure 2.

Identification and characterization of Mfa2 targeted mutants. (A) Mating assays of transformants targeted at the target 1 region. At the left is the mating assay of candidate Mfa2 mutants paring with wild type JG36. The fluffy colonies are the result of successful mating, while the dense colonies indicate a fail in mating. At the right are microscopic images of both colonies: yeast-like sporidia (mating negative) and filamentous hyphae (mating positive). Scale bars indicate 1 μm. The negative control is haploid JG35 with yeast-like colony, and the positive control is a mix of JG35/JG36 with filamentous hyphae. (B) PCR detection of the insert arms at the cleavage site. The left image was for the forward insertion and the right one for reverse insertion. For forward insertion, the sizes of PCR products were 1133 bp with primer pair mfa2C1F/CasR01 and 1148 bp with primer pair mfa2C1R/HygR01. For reverse insertion, the sizes of PCR products were 1030 bp by primers mfa2C1F/HygR01 and 1251 bp by primers mfa2C1R/CasR01. (C) Sequences of the Mfa2 flanking the insert at target 1 locus of the transformants. Underlined are RB and LB regions. The insertion regions are marked with wavy line. As could been seen, in both forward or reverse insertions, the whole RB and a small portion of the cassette at the 5′ end were lost, but only 9–13 LB nucleotides at the far most 3′ end were lost.