Fig. 4

CRISPR-Ca12a mediated gene deletion and replacement in Z. mobilis. The scheme of CRISPR–Cas12a mediated gene deletion (a) and replacement (b). Co-expression of Cas12a and crRNA that targets the Z. mobilis chromosome introduced double strand break (DSB). Donor plasmid contains the desired edit as well as additional homologous sequence flanking upstream and downstream of the target locus. Transformants generated from CRISPR–Cas12a assisted genome recombination were verified by colony PCR. For deletion of ZMO0028, 2.3 kb PCR fragment generated by primer pair 0028check-F/R indicates the wild-type genotype, whereas the presence of 1.5 kb PCR fragment indicate the recombinant genotype. For deletion of ZMO0347, 2.8 kb PCR fragment generated by primer pair 0347check-F/R represents the wild-type genotype, whereas the presence of 2.1 kb PCR fragment indicates the recombinant genotype. For mCherry insertion, 1.5 kb PCR fragment generated by primer pair 0028check-F/R indicates ZMO0028 mutant genotype, whereas the presence of 2.5 kb PCR fragment indicates the recombinant genotype. The insertion of LdhBc was identified by primer pair 0028check-F/R. A 2.3 kb PCR fragment indicates the wild-type genotype, while a 2.9 kb PCR fragment indicates the insertion of LdhBc. M represents DNA marker (DL5,000), 1 and 2 represents PCR pduducts from recombiant and wild-type strains, respectively (c), and the representative recombinants were then confirmed by Sanger sequencing (d). c.f.u and editing efficiency of Cas12-assisted in-frame deletions (e), c.f.u and editing efficiency of Cas12-assisted mCherry and LdhBc insertion (f). A mutant with endogenous gene ZMO0028 replaced by the reporter gene mCherry was also verified by flow cytometry, gray peak was signal from negative control ZM4 and pink peak from mutant ZM-mCherry (g). Values are the means of experiments with three or more technical replicates, error bars are standard deviation. HA homologous arm