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Fig. 5 | Microbial Cell Factories

Fig. 5

From: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

Fig. 5

Double-locus editing in in C. glutamicum. a Schematic representation of pCas9 and pgRNA7 which were used for ssDNA-directed recombineering. b Mutants verification by sequencing of rfp and rpsL genes. Plasmid pgRNA7 that simultaneously expressed two gRNAs targeting rfp and rpsL was first transformed into C. glutamicum ATCC 13032::rfp. The resultant strain was then induced to express RecT and co-transformed with pCas9 and two kinds of synthetic ssDNAs (10 μg rfp-off1 and 10 μg rpsL-K43R, Additional file 1: Table S1). After recovery, cells were spread on SGY plates supplemented with Km, Cm, and IPTG (0.01 mM) to induce Cas9 expression for counter-selection. Colonies were picked and verified by gene sequencing

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