Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a

Abstract

Background Functionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci. Results Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase. Conclusions We conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in Shigella flexneri by Cytosine Base Editing

“…We did observe a significant effect on the levels of icsB transcription ( Fig. S4D ), particularly for the S. flexneri icsB ( Q32X ) mutant, which is consistent with our previous report ( 42 ) that some mutations, including premature stop codons, can decrease mRNA levels without affecting observed phenotypes. Altogether, these results showed that base-edited premature stop codon mutations did not result in any observable polar effects, at least at the phenotype level.…”

“…Total RNA extraction and qPCR were performed as previously described, with minor modifications ( 42 , 59 ). Bacteria were grown similarly to the mutagenesis experiment, but instead of spotting the dilutions, the cultures were treated with ice-cold stop solution (95% [vol/vol] ethanol, 5% [vol/vol] phenol, pH 4) for 30 min on ice.…”

CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid inShigella flexneriby cytosine base editing

“…We did observe a significant effect on the levels of icsB transcription (Supplementary Fig. S4D), particularly for the S. flexneri icsB(Q32X) mutant, which is consistent with our previous report (42) that some mutations, including premature STOP codons, can decrease mRNA levels without affecting observed phenotypes.…”

“…We did observe a significant effect on the levels of icsB transcription (Supplementary Fig. S4D), particularly for the S. flexneri icsB(Q32X) mutant, which is consistent with our previous report (42) that some mutations, including premature STOP codons, can decrease mRNA levels without affecting observed phenotypes. Altogether, these results showed that base-edited premature STOP codon mutations did not result in any observable polar effects, at least at the phenotype level.…”

“…T otal RNA extraction and qPCR were performed as previously described, with minor modifications (42, 60). Bacteria were grown similarly to the mutagenesis experiment but instead of spotting the dilutions, the cultures were treated with ice-cold Stop Solution (95% v/v ml of ethanol, 5% v/v ml of phenol pH 4) for 30 min on ice.…”