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 (Knr, 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, Knr 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.