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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
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.
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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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