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A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to...

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Autores principales: Ellis, Nicole A., Kim, Byoungkwan, Tung, Jessica, Machner, Matthias P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862264/
https://www.ncbi.nlm.nih.gov/pubmed/33542442
http://dx.doi.org/10.1038/s42003-021-01672-7
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author Ellis, Nicole A.
Kim, Byoungkwan
Tung, Jessica
Machner, Matthias P.
author_facet Ellis, Nicole A.
Kim, Byoungkwan
Tung, Jessica
Machner, Matthias P.
author_sort Ellis, Nicole A.
collection PubMed
description Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.
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spelling pubmed-78622642021-02-11 A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila Ellis, Nicole A. Kim, Byoungkwan Tung, Jessica Machner, Matthias P. Commun Biol Article Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens. Nature Publishing Group UK 2021-02-04 /pmc/articles/PMC7862264/ /pubmed/33542442 http://dx.doi.org/10.1038/s42003-021-01672-7 Text en © This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021 Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ellis, Nicole A.
Kim, Byoungkwan
Tung, Jessica
Machner, Matthias P.
A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title_full A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title_fullStr A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title_full_unstemmed A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title_short A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila
title_sort multiplex crispr interference tool for virulence gene interrogation in legionella pneumophila
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862264/
https://www.ncbi.nlm.nih.gov/pubmed/33542442
http://dx.doi.org/10.1038/s42003-021-01672-7
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