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.

The Candida genus encompasses a diverse group of ascomycete fungi that have captured the attention of the scientific community, due to both their role in pathogenesis and emerging applications in biotechnology; the development of gene editing tools such as CRISPR, to analyze fungal genetics and perform functional genomic studies in these organisms, is essential to fully understand and exploit this genus, to further advance antifungal drug discovery and industrial value. However, genetic manipulation of Candida species has been met with several distinctive barriers to progress, such as unconventional codon usage in some species, as well as the absence of a complete sexual cycle in its diploid members. Despite these challenges, the last few decades have witnessed an expansion of the Candida genetic toolbox, allowing for diverse genome editing applications that range from introducing a single point mutation to generating large-scale mutant libraries for functional genomic studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is among the most recent of these advancements, bringing unparalleled versatility and precision to genetic manipulation of Candida species. Since its initial applications in Candida albicans, CRISPR-Cas9 platforms are rapidly evolving to permit efficient gene editing in other members of the genus. The technology has proven useful in elucidating the pathogenesis and host-pathogen interactions of medically relevant Candida species, and has led to novel insights on antifungal drug susceptibility and resistance, as well as innovative treatment strategies. CRISPR-Cas9 tools have also been exploited to uncover potential applications of Candida species in industrial contexts. This review is intended to provide a historical overview of genetic approaches used to study the Candida genus and to discuss the state of the art of CRISPR-based genetic manipulation of Candida species, highlighting its contributions to deciphering the biology of this genus, as well as providing perspectives for the future of Candida genetics.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug susceptibility and contribute towards the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

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Catheter-associated urinary tract infections (CAUTIs) account for 40% of hospital-acquired infections (HAIs). As 20 to 50% of hospitalized patients receive catheters, CAUTIs are one of the most common HAIs, resulting in increased morbidity, mortality, and health care costs. Candida albicans is the second most common CAUTI uropathogen, yet relative to its bacterial counterparts, little is known about how fungal CAUTIs are established. Here, we show that the catheterized bladder environment induces Efg1- and fibrinogen (Fg)–dependent biofilm formation that results in CAUTI. In addition, we identify the adhesin Als1 as the critical fungal factor for C. albicans Fg-urine biofilm formation. Furthermore, we show that in the catheterized bladder, a dynamic and open system, both filamentation and attachment are required, but each by themselves are not sufficient for infection. Our study unveils the mechanisms required for fungal CAUTI establishment, which may aid in the development of future therapies to prevent these infections.