Fig. 2. CRISPR/Cpf1 mediated homologous recombination in C. glutamicum . (A) The double target sites were located 243 bp and 683 bp from the start codon of the ldhA gene, respectively. The first homologous recombination occurred on the LdhAp region at the first heat shock, and the second crossing-over on the LdhAt region occurred after the second heat shock. (B) The deletion of the ldhA gene and simultaneous Knr gene insertion were confirmed via PCR analysis (PCR 1 and 2). (C) Confirmation of the complete inhibition of lactic acid production in the ldhA gene deletion mutant (ΔldhA-6 ). Glucose was used as the sole carbon source.