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Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a

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 hypo...

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Autores principales: Graffeuil, Antoine, Guerrero-Castro, Julio, Assefa, Aster, Uhlin, Bernt Eric, Cisneros, David A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9277811/
https://www.ncbi.nlm.nih.gov/pubmed/35831865
http://dx.doi.org/10.1186/s12934-022-01844-y
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author Graffeuil, Antoine
Guerrero-Castro, Julio
Assefa, Aster
Uhlin, Bernt Eric
Cisneros, David A.
author_facet Graffeuil, Antoine
Guerrero-Castro, Julio
Assefa, Aster
Uhlin, Bernt Eric
Cisneros, David A.
author_sort Graffeuil, Antoine
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01844-y.
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spelling pubmed-92778112022-07-14 Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a Graffeuil, Antoine Guerrero-Castro, Julio Assefa, Aster Uhlin, Bernt Eric Cisneros, David A. Microb Cell Fact Methodology 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01844-y. BioMed Central 2022-07-13 /pmc/articles/PMC9277811/ /pubmed/35831865 http://dx.doi.org/10.1186/s12934-022-01844-y Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Methodology
Graffeuil, Antoine
Guerrero-Castro, Julio
Assefa, Aster
Uhlin, Bernt Eric
Cisneros, David A.
Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title_full Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title_fullStr Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title_full_unstemmed Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title_short Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
title_sort polar mutagenesis of polycistronic bacterial transcriptional units using cas12a
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9277811/
https://www.ncbi.nlm.nih.gov/pubmed/35831865
http://dx.doi.org/10.1186/s12934-022-01844-y
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