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Characterizing a thermostable Cas9 for bacterial genome editing and silencing
CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineer...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698299/ https://www.ncbi.nlm.nih.gov/pubmed/29162801 http://dx.doi.org/10.1038/s41467-017-01591-4 |
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author | Mougiakos, Ioannis Mohanraju, Prarthana Bosma, Elleke F. Vrouwe, Valentijn Finger Bou, Max Naduthodi, Mihris I. S. Gussak, Alex Brinkman, Rudolf B. L. van Kranenburg, Richard van der Oost, John |
author_facet | Mougiakos, Ioannis Mohanraju, Prarthana Bosma, Elleke F. Vrouwe, Valentijn Finger Bou, Max Naduthodi, Mihris I. S. Gussak, Alex Brinkman, Rudolf B. L. van Kranenburg, Richard van der Oost, John |
author_sort | Mougiakos, Ioannis |
collection | PubMed |
description | CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range. |
format | Online Article Text |
id | pubmed-5698299 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-56982992017-11-24 Characterizing a thermostable Cas9 for bacterial genome editing and silencing Mougiakos, Ioannis Mohanraju, Prarthana Bosma, Elleke F. Vrouwe, Valentijn Finger Bou, Max Naduthodi, Mihris I. S. Gussak, Alex Brinkman, Rudolf B. L. van Kranenburg, Richard van der Oost, John Nat Commun Article CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range. Nature Publishing Group UK 2017-11-21 /pmc/articles/PMC5698299/ /pubmed/29162801 http://dx.doi.org/10.1038/s41467-017-01591-4 Text en © The Author(s) 2017 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 Mougiakos, Ioannis Mohanraju, Prarthana Bosma, Elleke F. Vrouwe, Valentijn Finger Bou, Max Naduthodi, Mihris I. S. Gussak, Alex Brinkman, Rudolf B. L. van Kranenburg, Richard van der Oost, John Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title | Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title_full | Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title_fullStr | Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title_full_unstemmed | Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title_short | Characterizing a thermostable Cas9 for bacterial genome editing and silencing |
title_sort | characterizing a thermostable cas9 for bacterial genome editing and silencing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698299/ https://www.ncbi.nlm.nih.gov/pubmed/29162801 http://dx.doi.org/10.1038/s41467-017-01591-4 |
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