FIGURE 3.

Replacement of rifZ by an antibiotic cassette with CRISPR-Cas12a-assisted precise cutting and HDR-mediated precise recombination in A. mediterranei U32. (A) Schematic of rifZ deletion. With the existence of a crRNA targeting rifZ, Cas12a introduced DSBs within rifZ gene, which could then be precisely repaired by a donor DNA fragment containing an apramycin resistance cassette and the flanking upstream and downstream homologous sequences. (B) The number of transformants obtained with three different crRNAs targeting different loci within rifZ. Plasmid pULcrRNA, which contained no crRNA guide sequences, was employed as a control plasmid. The data were obtained from three independent transformation experiments. (C) PCR amplification analysis of rifZ mutant with paired primers of rifZ-KO-F and rifZ-KO-R. The amplicon of the wild type was 4.8 kb in length, while was smaller in rifZ mutants. M, GeneRuler 1 kb DNA Ladder (Thermo Scientific); lanes 1 and 2, colonies obtained with pULrifZ1-LAR; lanes 3 and 4, colonies obtained with pULrifZ2-LAR; lanes 5–8, colonies obtained with pULrifZ3-LAR. (D) Sanger sequencing results of the amplicon from Figure 3C. All eight amplicons were sequenced and found to be correct, and only the results of the first colony were presented.