Fig. 4

The diagram of genomic editing procedure. a Day 1–3: The recombinant plasmid construction for targeted gene X in E. coli DH5α. Day 4–6, electroporate the plasmid into the electrocompetent cell of C. glutamicum and cultured for 2–3 days at 28 °C, examine the colonies using colony PCR method. N20 sequence at 5′-end of sgRNA was complementary to the targeted gene X, which guide Cas9 to break genome at PAM (NGG) site, the upstream and downstream homologous sequence in plasmid was used to repair the DSBs. Day 7–9, cure the plasmids in positive cells. If continuous editing was needed, make electrocompetent cells of the positive cells, electroporate another plasmid into it for the next editing cycle