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Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically atte...

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Autores principales: Collias, Daphne, Vialetto, Elena, Yu, Jiaqi, Co, Khoa, Almási, Éva d. H., Rüttiger, Ann-Sophie, Achmedov, Tatjana, Strowig, Till, Beisel, Chase L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9908933/
https://www.ncbi.nlm.nih.gov/pubmed/36754958
http://dx.doi.org/10.1038/s41467-023-36283-9
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author Collias, Daphne
Vialetto, Elena
Yu, Jiaqi
Co, Khoa
Almási, Éva d. H.
Rüttiger, Ann-Sophie
Achmedov, Tatjana
Strowig, Till
Beisel, Chase L.
author_facet Collias, Daphne
Vialetto, Elena
Yu, Jiaqi
Co, Khoa
Almási, Éva d. H.
Rüttiger, Ann-Sophie
Achmedov, Tatjana
Strowig, Till
Beisel, Chase L.
author_sort Collias, Daphne
collection PubMed
description Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.
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spelling pubmed-99089332023-02-10 Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria Collias, Daphne Vialetto, Elena Yu, Jiaqi Co, Khoa Almási, Éva d. H. Rüttiger, Ann-Sophie Achmedov, Tatjana Strowig, Till Beisel, Chase L. Nat Commun Article Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria. Nature Publishing Group UK 2023-02-08 /pmc/articles/PMC9908933/ /pubmed/36754958 http://dx.doi.org/10.1038/s41467-023-36283-9 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/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/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Collias, Daphne
Vialetto, Elena
Yu, Jiaqi
Co, Khoa
Almási, Éva d. H.
Rüttiger, Ann-Sophie
Achmedov, Tatjana
Strowig, Till
Beisel, Chase L.
Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title_full Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title_fullStr Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title_full_unstemmed Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title_short Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
title_sort systematically attenuating dna targeting enables crispr-driven editing in bacteria
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9908933/
https://www.ncbi.nlm.nih.gov/pubmed/36754958
http://dx.doi.org/10.1038/s41467-023-36283-9
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