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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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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. |
format | Online Article Text |
id | pubmed-9908933 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
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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