Polar mutagenesis of bacterial transcriptional units using Cas12a

Abstract

Bacterial genes are often organized in functionally related transcriptional units or operons. One such example is the fimAICDFGH operon, which codes for type I fimbriae in Escherichia coli. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the fim operon. Cas12a-mediated engineering of a terminator sequence inside the fimA 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 fim locus, had decreased mRNA levels of fimA, suggesting that the regulation of the fim 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 fimA. We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in E. coli, this opens the possibility of inhibiting competing metabolic routes.