“Polar mutagenesis of bacterial transcriptional units using Cas12a”

Bacterial genes are often organized in functionally related transcriptional units or operons. One such example is the operon, which codes for type I fimbriae in . We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the operon. Cas12a-mediated engineering of a terminator sequence inside the gene occurred with efficiencies between 10 and 30%, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Our results showed that some of the obtained mutants, including one with a single base substitution at the locus, had decreased mRNA levels of , suggesting that the regulation of the operon was disrupted. We corroborated the polar effect of these mutants by phenotypic assays and quantitative PCR, showing up to a 43 fold decrease in expression of genes downstream . We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in , this opens the possibility of inhibiting competing metabolic routes.