Fig. 3

Genome editing with CRISPR/Cas12a in Clostridium sp. AWRP. (A) Construction of CRISPR/Cas12a genome-editing plasmids that differ in the promoters driving Cas12a and crRNA expression. The combinations of two promoters were assembled in BamHI/XbaI-digested pKLJM343. Bidirectional transcriptional terminators (red arrows) of Clostridium acetobutylicum were cloned to prevent possible transcriptional interference between the elements on the plasmid. The table on the right shows the results of transformation and genome editing upon the introduction of various plasmid constructs. The plus (+) signs in the table indicate the occurrence of transformed colonies, and the minus signs (–) indicate no transformed colonies were observed. The numbers in brackets indicate the plasmid number (see Table S1). (B) Schematic diagram of the xylB deletion. Primer binding sites are indicated by red, half arrows. LA and RA indicate two homologous arms for the disruption of xylB. Primer sequences are available in Table S2. (C) Confirmation of the xylB mutant by colony PCR (top) and growth on LBFX medium (bottom). LBFX medium was prepared the same as LBFA medium except that 5 g L^− 1 fructose was replaced with 5 g L^− 1 xylose