Fig. 1

The design and stability of DRs-involved paired-gRNA plasmids pDG-A-X in E. coli. a The modular construction strategy of pDG-A-X series. pKB plasmid was used for PCR amplification of DNA part 1, which contained pDG-A-X backbone, one constitutive promoter J23119, and a gRNA scaffold. pKI plasmid was used for PCR amplification of part 2 series, which contained a gRNA fragment followed by another promoter J23119 and 20-bp space sequence. For the PCR reaction, the 20-bp space sequences specific for two targeted loci and the 20-bp overlap sequences for assembly were embedded in primers as a part of insert. Gibson Assembly method was used to assemble these parts into pDG-A-X series. b Representative PCR results for the deletion frequencies of pDG-A-100K after re-transformation. c The double restriction enzyme digestion analyses of pDG-A-100K and its deletion derivatives. d Effects of transformation methods and culture media on plasmid deletion frequency. e The plasmid deletion frequencies of pDG-A-100K after the re-transformation into various strains of E. coli. Values and error bars represent the mean and the s.d. (n = 3)