Fig. 5

Point mutation and gene insertion mediated by the CRISPR/Cas9 system in C. glutamicum ATCC 13032. a Schematic depicting the procedure for generating point mutations. 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 point mutation site was designed in the primers FR and RF. CF and CR are primers for PCR validation of editing efficiency. The SEQ primer is used for sequencing. b Schematic depicting the procedure for gene insertion. The 5′ end of the LR contains a 10 bp overhang region of the 5′ end of GFP. The 5′ end of GFPF contains a 10 bp overhang region of the 3′ end of the left arm. The 5′ end of GFPR 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 GFP arm. c The point mutation mediated by CRISPR/Cas9 in C. glutamicum ATCC 13032. The mutation efficiency was 6/6 and was confirmed by sequencing. d Gene insertion mediated by CRISPR/Cas9 in C. glutamicum ATCC 13032. The porB gene was replaced by the gfp gene. The efficiency for gfp insertion was 8/12, confirmed by PCR and sequencing