Fig. 1. Construction of the vector for CRISPR/Cpf1 genome editing inC. glutamicum . (A) pJYS3_Amp_MCS vector derived from pJYS3_ ΔcrtYF was constructed. (B) A double guide crRNAs set was incorporated into the pJYS3_Amp_MCS plasmid, between the HindIII and Xba1 restriction enzyme sites. (C, D) Homologous arms with a selection marker gene (Kn^r, kanamycin resistance gene) and co-expression cassette (Psod:sucE-Pro4:rpsLm) were finally inserted into the JYS3_Amp_DT plasmid between the Xma1 and Apa1 restriction enzyme sites. T1 and T2, target DNA sites on the ldhA gene of C. glutamicum ; Pro1, AmpR promoter; Pro2, PlacM promoter; Pro3, J23119 promoter; Pro4, Kn^r promoter derived from pJYS3_ ΔcrtYF; Psod, promoter; rmB T1 term and sacB T1 term, terminator regions of the rmB and sacB genes, respectively; sT1, sacB T1 terminator; LdhAp, lactate dehydrogenase 1 (ldhA ) promoter region; ldhAt, terminator region of ldhA gene.