Fig. 3

The impact of crRNA on lagging and leading strand as well as the length of direct repeats in artificial CRISPR arrays on transformation and editing efficiencies of the CRISPR–Cas12a assisted ssDNA recombineering in Z. mobilis. c.f.u and editing efficiency of ldh after editing by 59 nt template oligonucleotide targeting the lagging strand and leading strand, respectively (a), or by oligonucleotide template with different length targeting the lagging strand mediated by plasmid pEZ-sgr-ldh expressing ldh targeting crRNA (b). Transformants generated from CRISPR–Cas12a assisted ssDNA recombination targeting on lagging strand, leading strand (c), or oligonucleotide template with different length targeting the lagging strand (d). Transformants were screened by colony PCR with primer pair Ldh-check-F/R, and the PCR product was then digested with a PstI to investigate the recombination efficiency. A 2.1 kb fragment indicates wild-type genotype, whereas the presence of 1.2 and 0.9 kb fragments indicate recombinant genotypes. The representative ldh recombinants were confirmed by Sanger sequencing (e). A multiple comparison between groups was performed for the editing efficiencies using different length of ssDNA through analysis of variance (ANOVA) followed by unpaired two-tailed student-t test using GraphPad InStat software (GraphPad, Prism 8). P value < 0.05 was considered to be statistically significant, and ns represents non-significant. Values are the means of experiment with three or more technical replicates, error bars are standard deviation