Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression i...

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Autores principales:	Elvira Román, Ioana Coman, Daniel Prieto, Rebeca Alonso-Monge, Jesús Pla
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR Candida albicans Gal4 RNA scaffold catalase gene activation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4fc64b16a4ec417390ec6a68c5afc03b
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spelling	oai:doaj.org-article:4fc64b16a4ec417390ec6a68c5afc03b2021-11-15T15:22:04ZImplementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>10.1128/mSphere.00001-192379-5042https://doaj.org/article/4fc64b16a4ec417390ec6a68c5afc03b2019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00001-19https://doaj.org/toc/2379-5042ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast Candida albicans. By fusing an allele of Streptococcus pyogenes Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene CAT1, encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of CAT1 controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen. IMPORTANCE CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in Candida albicans using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.Elvira RománIoana ComanDaniel PrietoRebeca Alonso-MongeJesús PlaAmerican Society for MicrobiologyarticleCRISPRCandida albicansGal4RNA scaffoldcatalasegene activationMicrobiologyQR1-502ENmSphere, Vol 4, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Candida albicans Gal4 RNA scaffold catalase gene activation Microbiology QR1-502
spellingShingle	CRISPR Candida albicans Gal4 RNA scaffold catalase gene activation Microbiology QR1-502 Elvira Román Ioana Coman Daniel Prieto Rebeca Alonso-Monge Jesús Pla Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
description	ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast Candida albicans. By fusing an allele of Streptococcus pyogenes Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene CAT1, encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of CAT1 controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen. IMPORTANCE CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in Candida albicans using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.
format	article
author	Elvira Román Ioana Coman Daniel Prieto Rebeca Alonso-Monge Jesús Pla
author_facet	Elvira Román Ioana Coman Daniel Prieto Rebeca Alonso-Monge Jesús Pla
author_sort	Elvira Román
title	Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
title_short	Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
title_full	Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
title_fullStr	Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
title_full_unstemmed	Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>
title_sort	implementation of a crispr-based system for gene regulation in <italic toggle="yes">candida albicans</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/4fc64b16a4ec417390ec6a68c5afc03b
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Implementation of a CRISPR-Based System for Gene Regulation in <italic toggle="yes">Candida albicans</italic>

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