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Fig. 1 | Microbial Cell Factories

Fig. 1

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

Fig. 1

Optimization of CRISPR–Cas9 system by integrating cas9 gene into the chromosome of C. glutamicum. a A chromosome-borne Cas9 expression cassette for lethality-based selection via sgRNA. The fragments of Cas9 gene under the control of different promotors and RBSs were integrated into the genome using a series of pIN-cas9 plasmids by two rounds of homologous recombination, respectively. psgRNA upp harboring the sgRNA expression cassette was transformed into the Cas9-expressing strains by electroporation to detect lethality rate. b The numbers of transformants generated by electrotransformation of psgRNA upp into WT, WT::P hom -cas9, WT::P tuf -cas9, WT::P tuf -rbs1-cas9 and WT::P tuf -rbs2-cas9 strains. pXMJ19ts was used as a control. c The relative transcription level of cas9 gene. The transcription level of cas9 in WT::P tuf -cas9 was compared against that of cas9 in WT::P hom -cas9. d The growth curves of C. glutamicum strains harboring different cas9 expression cassettes. e The expression level of Cas9 protein detected by Western blotting. The expression of plasmid-borne Cas9 with ITPG induction was used as a positive control. f The numbers of transformants (red column) and editing efficiencies (blue column) generated by electrotransformation of pHAsgRNA upp into C. glutamicum strains harboring different cas9 expression cassettes. 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. g Verification of inactivation of uracil phosphoribosyltransferase. The upp-deficient mutant was capable of growing on CGX agar plates containing 5-FU (20 μM)

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