Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision

ABSTRACT We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ab...

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Autores principales:	Manning Y. Huang, Aaron P. Mitchell
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR biotechnology genetics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/61a963a5a31b4f6fbb8253a54216a264
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spelling	oai:doaj.org-article:61a963a5a31b4f6fbb8253a54216a2642021-11-15T15:21:45ZMarker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision10.1128/mSphere.00050-172379-5042https://doaj.org/article/61a963a5a31b4f6fbb8253a54216a2642017-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00050-17https://doaj.org/toc/2379-5042ABSTRACT We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.Manning Y. HuangAaron P. MitchellAmerican Society for MicrobiologyarticleCRISPRbiotechnologygeneticsMicrobiologyQR1-502ENmSphere, Vol 2, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR biotechnology genetics Microbiology QR1-502
spellingShingle	CRISPR biotechnology genetics Microbiology QR1-502 Manning Y. Huang Aaron P. Mitchell Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
description	ABSTRACT We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.
format	article
author	Manning Y. Huang Aaron P. Mitchell
author_facet	Manning Y. Huang Aaron P. Mitchell
author_sort	Manning Y. Huang
title	Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
title_short	Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
title_full	Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
title_fullStr	Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
title_full_unstemmed	Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision
title_sort	marker recycling in <named-content content-type="genus-species">candida albicans</named-content> through crispr-cas9-induced marker excision
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/61a963a5a31b4f6fbb8253a54216a264
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Marker Recycling in <named-content content-type="genus-species">Candida albicans</named-content> through CRISPR-Cas9-Induced Marker Excision

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