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Fig. 2 | Microbial Cell Factories

Fig. 2

From: A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

Fig. 2

RecET-assisted CRISPR–Cas9 genome editing in C. glutamicum. a A flow-chart for a single-plasmid-based and RecET-assisted CRISPR–Cas9 editing method in C. glutamicum. RecET gene under control of a propionate-inducible promoter P prp was integrated into WT::P tuf -rbs2-cas9 via a series of pIN-recET plasmid by two rounds of homologous recombination. pHAsgRNA was transformed by electroporation into the strain harboring cas9recET expression cassettes. After the recovery in BHIS supplemented with 0.5 g/L sodium propionate (PRP) at 30 °C for 2 h, cells were spread on chloramphenicol-resistant BHIS plate for 2-day cultivation. The positive colonies screened by colony PCR were subcultured in chloramphenicol-free BHIS medium at 37 °C overnight for plasmid curing. The edited strain without pHAsgRNA plasmids could be used for the next round of genome editing. Finally, the pOUT-cas9recET carrying two homologous arms was transformed into the edited strain to plug out the chromosomal cas9recET expression cassettes by two rounds of homologous recombination to get a final strain. b The number of transformants generated by electrotransformation of pHAsgRNA into WT::P tuf -rbs2-cas9::P prp -recET with or without propionate introduction. c Editing efficiencies under the noninducible or inducible RecET expression conditions. d The number of transformants generated by electrotransformation of pHAsgRNA into WT::P tuf -rbs2-cas9::P prp -rbs3-recET and WT::P tuf -rbs2-cas9::P prp -rbs4-recET strains. e Editing efficiencies in WT::P tuf -rbs2-cas9::P prp -rbs3-recET and WT::P tuf -rbs2-cas9::P prp -rbs4-recET strains. Significant differences in the data were determined using Student’s t-test (*P < 0.05, **P < 0.01). The data are derived from experiments performed at least three times, and the error bars represent the standard deviations

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