Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

Abstract Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and s...

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Autores principales:	Bjorn Thor Adalsteinsson, Thordis Kristjansdottir, William Merre, Alexandra Helleux, Julia Dusaucy, Mathilde Tourigny, Olafur Fridjonsson, Gudmundur Oli Hreggvidsson
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/71aa805bce4a4201afdca651fc406aba
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spelling	oai:doaj.org-article:71aa805bce4a4201afdca651fc406aba2021-12-02T16:51:49ZEfficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant10.1038/s41598-021-89029-22045-2322https://doaj.org/article/71aa805bce4a4201afdca651fc406aba2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89029-2https://doaj.org/toc/2045-2322Abstract Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5′-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.Bjorn Thor AdalsteinssonThordis KristjansdottirWilliam MerreAlexandra HelleuxJulia DusaucyMathilde TourignyOlafur FridjonssonGudmundur Oli HreggvidssonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bjorn Thor Adalsteinsson Thordis Kristjansdottir William Merre Alexandra Helleux Julia Dusaucy Mathilde Tourigny Olafur Fridjonsson Gudmundur Oli Hreggvidsson Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
description	Abstract Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5′-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.
format	article
author	Bjorn Thor Adalsteinsson Thordis Kristjansdottir William Merre Alexandra Helleux Julia Dusaucy Mathilde Tourigny Olafur Fridjonsson Gudmundur Oli Hreggvidsson
author_facet	Bjorn Thor Adalsteinsson Thordis Kristjansdottir William Merre Alexandra Helleux Julia Dusaucy Mathilde Tourigny Olafur Fridjonsson Gudmundur Oli Hreggvidsson
author_sort	Bjorn Thor Adalsteinsson
title	Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
title_short	Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
title_full	Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
title_fullStr	Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
title_full_unstemmed	Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant
title_sort	efficient genome editing of an extreme thermophile, thermus thermophilus, using a thermostable cas9 variant
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/71aa805bce4a4201afdca651fc406aba
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Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant

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