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Editor's Pick Research Article | Synthetic Biology

A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

Lauren Wensing, Jehoshua Sharma, Deeva Uthayakumar, Yannic Proteau, Alejandro Chavez, Rebecca S. Shapiro
Aaron P. Mitchell, Editor
Lauren Wensing
aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Jehoshua Sharma
aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Deeva Uthayakumar
aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Yannic Proteau
aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Alejandro Chavez
bDepartment of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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Rebecca S. Shapiro
aDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Aaron P. Mitchell
Carnegie Mellon University
Roles: Editor
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DOI: 10.1128/mSphere.00002-19
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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.

  • Copyright © 2019 Wensing et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans
Lauren Wensing, Jehoshua Sharma, Deeva Uthayakumar, Yannic Proteau, Alejandro Chavez, Rebecca S. Shapiro
mSphere Feb 2019, 4 (1) e00002-19; DOI: 10.1128/mSphere.00002-19

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A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans
Lauren Wensing, Jehoshua Sharma, Deeva Uthayakumar, Yannic Proteau, Alejandro Chavez, Rebecca S. Shapiro
mSphere Feb 2019, 4 (1) e00002-19; DOI: 10.1128/mSphere.00002-19
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KEYWORDS

CRISPR
CRISPRi
Candida
Candida albicans
fungal genetics
genetic regulation
genetic technology

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