Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>

ABSTRACT Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging oppo...

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Autores principales:	Emily L. Norton, Racquel K. Sherwood, Richard J. Bennett
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR DNA double-strand break homology-directed repair nonhomologous end joining Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/3028788c37514b9aa72ceec294da00a5
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spelling	oai:doaj.org-article:3028788c37514b9aa72ceec294da00a52021-11-15T15:21:46ZDevelopment of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>10.1128/mSphere.00217-172379-5042https://doaj.org/article/3028788c37514b9aa72ceec294da00a52017-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00217-17https://doaj.org/toc/2379-5042ABSTRACT Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.Emily L. NortonRacquel K. SherwoodRichard J. BennettAmerican Society for MicrobiologyarticleCRISPRDNA double-strand breakhomology-directed repairnonhomologous end joiningMicrobiologyQR1-502ENmSphere, Vol 2, Iss 3 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR DNA double-strand break homology-directed repair nonhomologous end joining Microbiology QR1-502
spellingShingle	CRISPR DNA double-strand break homology-directed repair nonhomologous end joining Microbiology QR1-502 Emily L. Norton Racquel K. Sherwood Richard J. Bennett Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
description	ABSTRACT Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.
format	article
author	Emily L. Norton Racquel K. Sherwood Richard J. Bennett
author_facet	Emily L. Norton Racquel K. Sherwood Richard J. Bennett
author_sort	Emily L. Norton
title	Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
title_short	Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
title_full	Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
title_fullStr	Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
title_full_unstemmed	Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>
title_sort	development of a crispr-cas9 system for efficient genome editing of <named-content content-type="genus-species">candida lusitaniae</named-content>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/3028788c37514b9aa72ceec294da00a5
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Development of a CRISPR-Cas9 System for Efficient Genome Editing of <named-content content-type="genus-species">Candida lusitaniae</named-content>

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