Fig. 1
Plasmid design and construction of the all-in-one plasmid CRISPR-Cas9 system. a Assembly of spacer and donar DNA. The gRNAtarget under the constitutive promoters for genome editing. The another gRNArep under the inducible promoter for plasmid curing. b Squence of gRNArep under the control of PacoR promoter. c Strategy for iterative genome editing in B. subtilis. In the genome editing phase, sgRNAtarget/Cas9 complex cuts the genome for homologous recombination. In the plasmid curing phase, sgRNArep/Cas9 complex tragets the repA gene to eliminate the editing plasmid. d Tetracycline sensitivity test for the plasmid self-curing system, 16 clones were picked for tetracycline sensitivity test, and only one single clone showed tetracycline resistance