A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

Lauren Wensing; Jehoshua Sharma; Deeva Uthayakumar; Yannic Proteau; Alejandro Chavez; Rebecca S Shapiro

doi:10.1128/mSphere.00002-19

A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans

mSphere. 2019 Feb 13;4(1):e00002-19. doi: 10.1128/mSphere.00002-19.

Authors

Lauren Wensing^#¹, Jehoshua Sharma^#¹, Deeva Uthayakumar¹, Yannic Proteau¹, Alejandro Chavez², Rebecca S Shapiro³

Affiliations

¹ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
² Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA.
³ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada shapiror@uoguelph.ca.

^# Contributed equally.

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.

Keywords: CRISPR; CRISPRi; Candida; Candida albicans; fungal genetics; genetic regulation; genetic technology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems*
Candida albicans / genetics*
Gene Expression Regulation, Fungal
Genetic Engineering / methods*
Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems / genetics
Transcription, Genetic*

Substances

RNA, Guide, CRISPR-Cas Systems
CRISPR-Associated Protein 9