Biology subjects

Gervais, N. C.

Publications and source records attributed to Gervais, N. C..

6 recordsLinked to original sources

bioRxiv2026

CRISPR-activation reveals key resistance genes and vulnerabilities of copy number variants in Candida albicans

Changes in gene copy number are an extremely important source of variation and are frequently observed during the acquisition of drug resistance. The opportunistic human fungal pathogen Candida albicans frequently adapts to antifungal drugs via large copy number variations (CNVs) that amplify hundreds of genes simultaneously. Despite the recurrent amplification of CNVs across diverse clinical isolates, the genes that contribute to drug resistance are not known. Additionally, by amplifying many genes, CNVs might result in cross-adaptation or fitness trade-offs to multiple environments, which has major implications for how CNVs are expected to contribute to adaptation in complex environments like a mammalian host. We use CRISPR-activation to systematically assay the fitness effects of individually overexpressing [~]800 genes in four genetically diverse isolates across eight physiologically relevant environments. We identify 198 genes with significant fitness effects in at least one environment in one or more genetic backgrounds. We identify novel genes with positive fitness effects in two different classes of antifungal drug and observe frequent gene-by-environment interactions for the fitness effects of gene overexpression. Additive fitness effects of individual gene overexpression are a significant predictor of the fitness of multiple isolates with CNVs and can explain fitness trade-offs observed between classes of antifungal drug for the CNV isolates. These findings identify genes that increase fitness in drug and those that create vulnerabilities in CNV isolates and can help inform treatment of isolates adapting to antifungal drug via CNVs.

microbiology↗

bioRxiv2026

Chromosome-scale CRISPR screening reveals secretory pathway genes as drivers of aneuploidy-mediated antifungal tolerance

The gain or loss of chromosomes in eukaryotes often drives aberrant phenotypes by altering the expression levels of hundreds or thousands of genes. In the case of beneficial aneuploidies, the genetic basis of fitness improvement has rarely been pinpointed, and identifying the causal genes remains a major challenge in engineering biology. The leading cause of human fungal infections, Candida albicans, frequently acquires extra copies of chromosome R (ChrR) following exposure to azole antifungal drugs, resulting in heightened antifungal tolerance. Here, we combine RNA-seq with parallel chromosome-wide CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens to systematically profile the ChrR genes contributing to azole tolerance. Using multiplexed CRISPR-dCas12a tools, we further characterize the combinatorial effects of candidate genes and uncover a central role for post-Golgi secretory trafficking in antifungal tolerance. Specifically, we demonstrate that the secretory pathway regulators SEC4 and YPT31 are both necessary and sufficient for ChrR-mediated azole tolerance. By leveraging a large-scale CRISPRa screen in a fungal pathogen, our work functionally dissects one of the most common aneuploidies observed in C. albicans, provides mechanistic insight into the molecular basis of antifungal tolerance, and establishes a generalizable framework for studying aneuploidy-mediated phenotypes across eukaryotic organisms.

microbiology↗

bioRxiv2025

The Unique Efg1 Fungal Virulence Regulon in the Catheterized Bladder Environment

Urinary catheterization, a common procedure in hospitals and nursing home facilities, is a primary driver of hospital-acquired infections (HAI). These devices frequently lead to catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans has emerged as the second most common causative agent of CAUTIs; yet, its pathogenesis is poorly understood, which complicates development of efficient treatments. Previously, we identified the transcription factor Efg1 as a critical virulence driver in C. albicans CAUTIs. However, its specific downstream targets within the unique bladder microenvironment remained unknown. This study identifies, for the first time, the complete Efg1 regulon that is active during growth in human urine. We confirmed the clinical relevance of this discovery, finding that many of these Efg1-regulated factors are present and significantly upregulated in catheter samples from patients with C. albicans infections. Furthermore, we characterized two of these key factors, ECE1 and EED1, validating their roles both in vitro in urine conditions and in vivo using a CAUTI mouse model. Identifying the tissue-specific downstream targets of Efg1 elucidates the precise mechanism of fungal CAUTI. This knowledge provides a new roadmap for developing targeted therapeutics, offering vital antimicrobial-sparing strategies to combat these life-threatening infections. SIGNIFICANCECatheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections that can lead to severe complications and death. Although most are caused by bacteria, the fungus Candida albicans is an increasingly prevalent cause, yet the pathogenesis of fungal CAUTIs is poorly understood. Previous research identified Efg1 as necessary for CAUTI, and now this study defines the urine-specific Efg1 regulon, validating its clinical relevance in catheter samples from infected patients. We further assessed how key downstream factors, Ece1 and Eed1, contribute to bladder infection. This first report of the urine-specific EFG1 network provides new targets for diagnosing and treating these life-threatening infections.

microbiology↗

bioRxiv2025

HyperdCas12a-Based Multiplexed Genetic Regulation in Candida albicans

Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPRa and CRISPRi tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.

genetics↗

bioRxiv2025

Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9

The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.

microbiology↗

bioRxiv2022

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

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 resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

microbiology↗