A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans Lauren Wensing, a Jehoshua Sharma, a Deeva Uthayakumar, a Yannic Proteau, a Alejandro Chavez, b Rebecca S. Shapiro a a Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada b Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA ABSTRACT Fungal pathogens are emerging as an important cause of human dis- ease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessi- tates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clus- tered 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 en- dogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demon- strated that fusion of dCas9 to an Mxi1 repressor domain was able to further en- hance 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 dis- ease and mortality, and Candida albicans is among the most common causes of fun- gal disease. Studying this important fungal pathogen requires a comprehensive ge- netic 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 interfer- ence (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 demon- strated 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 I nvasive fungal infections have emerged as an important cause of human mortality, particularly for an ever-increasing population of immunocompromised individuals (1–3). The rise in the incidence of these opportunistic invasive infections is associated with many factors, including the HIV/AIDS epidemic and the growing number of patients receiving immunosuppressive therapeutics for bone marrow and organ trans- plantations or for the treatment of autoimmune disorders (4). Invasive fungal infections are associated with disproportionately high rates of patient mortality (30% to 90% mortality, depending on the pathogen and patient group [5, 6]), and with a massive Citation Wensing L, Sharma J, Uthayakumar D, Proteau Y, Chavez A, Shapiro RS. 2019. A CRISPR interference platform for efficient genetic repression in Candida albicans. mSphere 4:e00002-19. https://doi.org/10.1128/mSphere .00002-19. Editor Aaron P. Mitchell, Carnegie Mellon University Copyright © 2019 Wensing et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Rebecca S. Shapiro, shapiror@uoguelph.ca. L.W. and J.S. contributed equally to this article. For a companion article on this topic, see https://doi.org/10.1128/mSphere.00001-19. Development of a new CRISPRi tool for genetic repression in Candida albicans, work by @ShapiroRebecca @UofG_MCB Received 4 January 2019 Accepted 18 January 2019 Published 13 February 2019 RESEARCH ARTICLE Synthetic Biology crossm January/February 2019 Volume 4 Issue 1 e00002-19 msphere.asm.org 1