Fig. 4

Construction of a double mutant of prophage clusters. (A) Schematic diagram of the iterative procedure of gene knockout with the CRISPR/Cas12 system. (B) Curing of pKLJM359 after deletion of the first prophage group (DMR38_15715-15570). The replica was done after three serial transfers on LBFA (2% inoculum) in the absence of antibiotic pressure. (C) Schematic diagram of two prophage groups. Two homologous arms (LA and RA) are shown in blue boxes, and the binding sites of the verification primers (F1 to F4 and R1 to R4) are indicated in half arrows, respectively. Primer sequences are available in Table S2. (D) Verification of genotype of the double deletion mutants. Three colonies were chosen after curing the second target plasmid. The blue arrows indicate the size of the amplified PCR products with F1/R1 (as indicated in C). The black arrow indicates a nonspecific PCR product from the wild-type genomic DNA amplified with F1/R1