Fig. 1

Overview of CRISPR/Cas9 genome editing strategy in Escherichia coli. The strain to be mutagenized [E. coli BL21(DE3)∆ompA] is first transformed with the pCasRed plasmid expressing the λ Red (Exo, Beta, Gam) machinery, the Cas9 endonuclease, and tracrRNA. Subsequently, the strain is co-transformed with pCRISPR-SacB-gDNA, and a synthetic, mutation-inducing oligonucleotide [donor DNA (dDNA)]. The pCRISPR-SacB-gDNA plasmid encodes the gRNA that specifies the site of cleavage and the endonuclease Cas9 recognizes the gRNA together with the tracrRNA, which anneals to gRNA forming a three-component complex. After the base pairing of gRNA to the target site, the Cas9 mediates the chromosomal DNA double strand break (upper panel). The double strand break is repaired by λ Red-mediated homologous recombination taking place between the extremities of the cleaved chromosomal DNA and the donor DNA (lower panel). For the sequence of constitutive promoters P1 and P2 see ADDGENE #4287 [9]; for sequence of constitutive promoter P3 see ADDGENE #42875 [9] and for P4 constitutive promoter sequence see ADDGENE #13036 [24]. For the arabinose-inducible promoter pBAD see pKOBEG plasmid [20]