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.