FIGURE 4.

Markerless deletion of rifZ gene with the Cas12a and HDR systems in U32. (A) Schematic of markerless deletion of rifZ gene. Cas12a-generated DSBs in rifZ could be repaired by HDR. (B) Verification of rifZ deletion mutants by colony PCR with paired primers of rifZ+P-F and rifZ+P-R. M, GeneRuler 1 kb DNA Ladder (Thermo Scientific). Lanes 1–4 represented rifZ-KO-1 to rifZ-KO-4, respectively. (C) Sanger sequencing results from the amplicon of rifZ-KO-1 in Figure 4B. (D) Schematic of the rifZ-KO-4 mutant in Figure 4B. The mutant was obtained via NHEJ-mediated repair of the Cas12a-generated DSBs in rifZ gene. Confirmed by Sanger sequencing, 692-bp sequences were found to be deleted at the Cas12a cleavage site. (E) The growth phenotype of rifZ mutants on minimal medium. Serially diluted liquid culture was spotted on plates and cultured at 30°C for 5 days, and only the wild type produced pigmented rifamycin SV.