Fig. 2

Gene deletion using CRISPR/Cas9 and plasmid-borne editing template in C. glutamicum SL4. a Schematic representation of pCas9, pgRNA3, and gene deletion event. b Transformants harboring pCas9 and pgRNA3 on a SGY plate supplemented with Km, Cm, and IPTG (1 mM). pCas9 and pgRNA3 were co-transformed into strain SL4 simultaneously and cells were spread on SGY plates with Km, Cm, and IPTG (1 mM) immediately after recovery. Colonies marked in red cycles were the so-called abnormally large colonies. c Schematic representation of different patterns of pCas9 mutation. Gene deletion, cas9 gene was removed from pCas9. Nonsense mutation, a T465G mutation occurred, generating a stop codon (TAT to TAG) in cas9 gene. Insertional inactivation, a transposase encoding gene from E. coli was inserted into cas9 gene, which deactivated cas9. d Colony counting and editing efficiency calculation of C. glutamicum SL4. Colonies on SGY plates supplemented with Km, Cm, and IPTG (1 mM) were counted and verified using colony PCR. e PCR verification of ldhA deletion in C. glutamicum SL4 using the primer pair (ldhA-up and ldhA-down) shown in a. M, DNA marker; –, wild-type control; 1–8, eight small colonies. This displayed the result of Replicate 1 in c. The results of Replicate 2 and Replicate 3 were shown in Additional file 1: Figure S2