Figure 1.

Characterization of a knockout cassette for rapid generation and screening. (A) Schematic of the transformation workflow and integration of the knockout cassette by homologous recombination. (B) Example of DNA gel electrophoresis of knockout fragment integration. PCR was performed on genomic DNA extracted from individual colonies after transformation. (C) Observations from simultaneous targeting of pep4 and prb1 with varied knockout fragment homology arm lengths. Each homology arm length represents one transformation. Genomic loci were evaluated by PCR and Sanger sequencing. (D) Integration of the knockout cassette at mig1 and mig2 with different sgRNAs. Genomic loci were assessed by PCR and gel electrophoresis. (E) Integration and knockout efficiencies of gut1 with varied homology arm lengths. Bars represent independent transformations, with all or a maximum of 16 colonies analyzed. (F) Rapid generation of gene-specific knockout fragments by PCR.