A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>

ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehe...

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Autores principales:	Lauren Wensing, Jehoshua Sharma, Deeva Uthayakumar, Yannic Proteau, Alejandro Chavez, Rebecca S. Shapiro
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR CRISPRi Candida Candida albicans fungal genetics genetic regulation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/156f6fda551b49039cbdcd1389eb35ac
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spelling	oai:doaj.org-article:156f6fda551b49039cbdcd1389eb35ac2021-11-15T15:22:05ZA CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>10.1128/mSphere.00002-192379-5042https://doaj.org/article/156f6fda551b49039cbdcd1389eb35ac2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00002-19https://doaj.org/toc/2379-5042ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.Lauren WensingJehoshua SharmaDeeva UthayakumarYannic ProteauAlejandro ChavezRebecca S. ShapiroAmerican Society for MicrobiologyarticleCRISPRCRISPRiCandidaCandida albicansfungal geneticsgenetic regulationMicrobiologyQR1-502ENmSphere, Vol 4, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR CRISPRi Candida Candida albicans fungal genetics genetic regulation Microbiology QR1-502
spellingShingle	CRISPR CRISPRi Candida Candida albicans fungal genetics genetic regulation Microbiology QR1-502 Lauren Wensing Jehoshua Sharma Deeva Uthayakumar Yannic Proteau Alejandro Chavez Rebecca S. Shapiro A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
description	ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.
format	article
author	Lauren Wensing Jehoshua Sharma Deeva Uthayakumar Yannic Proteau Alejandro Chavez Rebecca S. Shapiro
author_facet	Lauren Wensing Jehoshua Sharma Deeva Uthayakumar Yannic Proteau Alejandro Chavez Rebecca S. Shapiro
author_sort	Lauren Wensing
title	A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
title_short	A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
title_full	A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
title_fullStr	A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
title_full_unstemmed	A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>
title_sort	crispr interference platform for efficient genetic repression in <italic toggle="yes">candida albicans</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/156f6fda551b49039cbdcd1389eb35ac
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A CRISPR Interference Platform for Efficient Genetic Repression in <italic toggle="yes">Candida albicans</italic>

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