Fig. 3
Gene disruption of three reported genes, Rei1, Ppg1, and Dbf2, via Mo_tRNAArg-gRNA-Cas9/pKOV21 system. a–c Strategy of gene replacement for genes, the sgRNA-guided Cas9-induced double-strand break (DSB) sites was indicated with scissor symbol. d–f Phenotype of 24 randomly selected G418-sensitive transformants of each gene disruption. g–i PCR verification using the Hyg_up/check_up and Hyg_down/check_down primers (as indicated in a–c) to confirm the correct replacement of each gene with Hyg. Ten random transformants with the correct knockout mutant phenotype of each gene were selected. In Fig. 3 h, the wild-type P131 and Δmoppg1-0 were used as negative and positive controls, respectively. j–l PCR detection for Cas9 insertion into genomes of the ten transformants of each gene mentioned in g–i. In Fig. 3g, i, the genomic DNA of the wild-type strain P131 and water were used as negative controls. The Mo_tRNA-gRNA-Cas9 vector was used as positive control. In Fig. 3k, Δmoppg1-0 was used as a negative control. m–o Colony phenotypes of each gene disruption strains and wild-type strain P131 cultured on OTA plates for 5 days