FIG 6.

sgRNA multiplexing for complete gene deletions. We introduced two sgRNA cassettes into the Cas9-expressing vector, targeting the 5′- and 3′-end of the VEA1 CDS, respectively. Colony PCR of primary transformants (lower part of panel A) resulted in two bands for 4 of 10 isolates and only one band for 2 isolates, indicating partial or complete loss of the VEA1 CDS in those mutants. (B) Southern hybridization of vea1∆ mutants confirmed the deletion of the VEA1 CDS in two independent mutants. Location of the probe is shown in the upper part of panel B, and the Southern blot is shown in the lower part of panel B. (C) Phenotypical characterization of the mutants grown at room temperature (RT) was performed by spotting dilutions of wild-type (G217B ura5⁻) and vea1∆ mutant strains on plates. The vea1∆ mutants displayed accelerated filamentation as compared to the parental strain as indicated by the increased density of fluffy white filamentous growth. The difference between wild-type and mutant strains was most pronounced after 6 days of incubation.