Fig. 2

CRISPR/Cas9-mediated genome editing in C. glutamicum ATCC 13032 and C. glutamicum CGMCC1.15647. a Schematic depicting editing procedures. The left and right arms are regions from the targeted gene and are amplified by PCR from C. glutamicum genomic DNA. LF and LR primers are used to amplify the left arm, and RF and RR primers are used to amplify the right arm. For Gibson assembly, the 5′ end of LF contains a 20 bp overhang region of the 5′ end of the BglII site from the pFST plasmid. The 5′ end of LR contains a 10 bp overhang region of the 5′ end of the right arm. The 5′ end of RF contains a 10 bp overhang region of the 3′ end of the left arm. The 5′ end of RR contains a 20 bp overhang region of the 3′ end of the BglII site from the pFST plasmid. CF and CR are primers for PCR validation of editing efficiency. The SEQ primer is used for sequencing. b The CRISPR/Cas9 system mediated disruption of the porB gene in C. glutamicum ATCC 13032. The editing efficiency was 18/18. The lane ‘ck’ is the PCR product from the wild-type strain. These results were confirmed by sequencing. c The CRISPR/Cas9 system mediated disruption of the proB gene in C. glutamicum CGMCC1.15647. The editing efficiency was 16/16