Fig. 1

Schematic overview of the CRISPR–Cpf1-based system for iterative genome editing in C. glutamicum. a Schematic representation of genome editing using an all-in-one plasmid. The pre-crRNA is processed by Cpf1 into a mature crRNA, which then recognizes the target sequence and PAM sequence. The Cpf1-crRNA complex binds to the target site and induces a staggered cut to generate a double-strand break (DSB). The plasmid-borne template repairs the DSB through homologous recombination. The successfully edited colonies can be verified by PCR using the F/R primers located outside of the homologous arms. b A schematic representation of the procedure for CRISPR-Cpf1-assisted genome editing in C. glutamicum. A plasmid containing Cpf1, crRNA, and the repair template and another plasmid containing Cpf1 and crRNA are constructed. The two plasmids are then transformed into competent C. glutamicum cells and the cultures are spread on BHIS plates containing kanamycin. The single colonies are cultivated in complete medium to extract genomic DNA for PCR and DNA sequencing. The plasmid can be cured by overnight incubation in kanamycin-free medium at 37 °C and the correctly edited strain is then obtained by verifying antibiotic sensitivity. If necessary, the obtained strain can be used for the next round of editing. c The control (left) and edited (right) strain on the plates. The left plate is without 5-fluorouracil (5-FU) and the right plate is supplemented with 100 μM 5-FU. Under normal circumstances, the colonies on the right plate will all be positive. d PCR validation of upp gene deletion. The primers, F17/R17, used for verification, are located outside of the homologous arms. The amplified fragments are 2814 bp (wild-type) and 2128 bp (edited strain)