Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds

The escalating prevalence of drug-resistant fungal infections and the emergence of intrinsically resistant fungal pathogens present a significant global health challenge. The evolutionary proximity between mammalian hosts and fungi complicates the development of novel antifungal agents, often leading to off-target toxicity. Consequently, antifungal drug discovery prioritizes fungal-specific proteins as potential therapeutic targets. Effective drug targets are characterized by their involvement in essential cellular processes and high sensitivity to inhibition. Gene repression strategies serve as valuable models for evaluating drug-mediated inhibition and establishing dosage-sensitivity profiles for specific genes. Traditional gene repression methods in Candida albicans, a prominent fungal pathogen, are often labor-intensive and limited in throughput and genetic background applicability. This

research introduces and adapts pooled CRISPR interference (CRISPRi) screening in C. albicans for the first time, enabling large-scale functional genomic analysis. Utilizing this high-throughput methodology, the study systematically assesses the repression sensitivity of over one hundred essential genes, which are conserved in fungi but absent in humans. The investigation successfully identifies numerous highly dosage-sensitive genes distributed across various cellular components and metabolic pathways, providing critical insights for the development of targeted antifungal therapies.

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