New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii. These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each...

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Autores principales:	Valmik K. Vyas, G. Guy Bushkin, Douglas A. Bernstein, Matthew A. Getz, Magdalena Sewastianik, M. Inmaculada Barrasa, David P. Bartel, Gerald R. Fink
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR Candida Naumovozyma Saccharomyces albicans castellii Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fc3e689ff07143b294c2db074a0e1a1e
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spelling	oai:doaj.org-article:fc3e689ff07143b294c2db074a0e1a1e2021-11-15T15:22:14ZNew CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi10.1128/mSphere.00154-182379-5042https://doaj.org/article/fc3e689ff07143b294c2db074a0e1a1e2018-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00154-18https://doaj.org/toc/2379-5042ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii. These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii. The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.Valmik K. VyasG. Guy BushkinDouglas A. BernsteinMatthew A. GetzMagdalena SewastianikM. Inmaculada BarrasaDavid P. BartelGerald R. FinkAmerican Society for MicrobiologyarticleCRISPRCandidaNaumovozymaSaccharomycesalbicanscastelliiMicrobiologyQR1-502ENmSphere, Vol 3, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Candida Naumovozyma Saccharomyces albicans castellii Microbiology QR1-502
spellingShingle	CRISPR Candida Naumovozyma Saccharomyces albicans castellii Microbiology QR1-502 Valmik K. Vyas G. Guy Bushkin Douglas A. Bernstein Matthew A. Getz Magdalena Sewastianik M. Inmaculada Barrasa David P. Bartel Gerald R. Fink New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
description	ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii. These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii. The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.
format	article
author	Valmik K. Vyas G. Guy Bushkin Douglas A. Bernstein Matthew A. Getz Magdalena Sewastianik M. Inmaculada Barrasa David P. Bartel Gerald R. Fink
author_facet	Valmik K. Vyas G. Guy Bushkin Douglas A. Bernstein Matthew A. Getz Magdalena Sewastianik M. Inmaculada Barrasa David P. Bartel Gerald R. Fink
author_sort	Valmik K. Vyas
title	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
title_short	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
title_full	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
title_fullStr	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
title_full_unstemmed	New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi
title_sort	new crispr mutagenesis strategies reveal variation in repair mechanisms among fungi
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/fc3e689ff07143b294c2db074a0e1a1e
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New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

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