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Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

[Image: see text] Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptoco...

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Autores principales: Mougiakos, Ioannis, Bosma, Elleke F., Weenink, Koen, Vossen, Eric, Goijvaerts, Kirsten, van der Oost, John, van Kranenburg, Richard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2017
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440800/
https://www.ncbi.nlm.nih.gov/pubmed/28146359
http://dx.doi.org/10.1021/acssynbio.6b00339
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author Mougiakos, Ioannis
Bosma, Elleke F.
Weenink, Koen
Vossen, Eric
Goijvaerts, Kirsten
van der Oost, John
van Kranenburg, Richard
author_facet Mougiakos, Ioannis
Bosma, Elleke F.
Weenink, Koen
Vossen, Eric
Goijvaerts, Kirsten
van der Oost, John
van Kranenburg, Richard
author_sort Mougiakos, Ioannis
collection PubMed
description [Image: see text] Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45–55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.
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spelling pubmed-54408002017-05-24 Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9 Mougiakos, Ioannis Bosma, Elleke F. Weenink, Koen Vossen, Eric Goijvaerts, Kirsten van der Oost, John van Kranenburg, Richard ACS Synth Biol [Image: see text] Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45–55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C. American Chemical Society 2017-02-01 2017-05-19 /pmc/articles/PMC5440800/ /pubmed/28146359 http://dx.doi.org/10.1021/acssynbio.6b00339 Text en Copyright © 2017 American Chemical Society This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License (http://pubs.acs.org/page/policy/authorchoice_ccbyncnd_termsofuse.html) , which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.
spellingShingle Mougiakos, Ioannis
Bosma, Elleke F.
Weenink, Koen
Vossen, Eric
Goijvaerts, Kirsten
van der Oost, John
van Kranenburg, Richard
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title_full Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title_fullStr Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title_full_unstemmed Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title_short Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
title_sort efficient genome editing of a facultative thermophile using mesophilic spcas9
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440800/
https://www.ncbi.nlm.nih.gov/pubmed/28146359
http://dx.doi.org/10.1021/acssynbio.6b00339
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