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

Wensing, L.; Despres, P.; Francis, D.; Fogal, M.; Hendriks, A.; Gervais, N.; Fikry, C.; Adamu Bukari, A.-R.; Gerstein, A.; Cuomo, C. A.; Shapiro, R. S.

doi:10.64898/2025.12.19.695556

bioRxiv · 10.64898/2025.12.19.695556

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

Wensing, L.·Despres, P.·Francis, D.·Fogal, M.·Hendriks, A.·Gervais, N.·Fikry, C.·Adamu Bukari, A.-R.·Gerstein, A.·Cuomo, C. A.·Shapiro, R. S.

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Abstract

The rising rate of drug-resistant fungal infections and the emergence of fungal pathogens with intrinsic resistance phenotypes are a growing concern. The close evolutionary distance between mammals and fungi complicates the design of new antifungals and increases the chances of toxic off-target effects. As such, antifungal drug development usually focuses on fungal-specific proteins when considering potential new targets. Ideal drug targets should mediate essential cell processes and be highly sensitive to inhibition. Targeted gene repression can serve as a model for drug-mediated inhibition and for determining the dosage-sensitivity profile of genes of interest. In the fungal pathogen Candida albicans, classical approaches for gene repression can be labour-intensive and limited to one genetic background due to low throughput. Here, we adapt pooled CRISPRi screening in C. albicans for the first time and exploit this technique for large-scale functional genomic analysis. Through pooled CRISPRi screening, we test the repression sensitivity of over a hundred essential genes conserved in fungi but absent in humans, and successfully identify highly dosage-sensitive genes across multiple cell components and pathways. By extending our analysis to ten diverse environmental conditions, we show how the environment influences dosage-sensitivity profiles. Finally, we extend our experiments to two clinical drug-resistant C. albicans strain backgrounds and demonstrate that many of the fitness defects we observed are conserved in resistant clinical isolates. Together, our results highlight a set of genes that are highly dosage-sensitive across different genetic and environmental contexts, making them attractive targets for further investigation. By facilitating rapid, efficient large-scale functional genomics assays across diverse genetic backgrounds, CRISPRi pooled screening will open new frontiers in C. albicans biology.

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Wensing, L., Despres, P., Francis, D., Fogal, M., Hendriks, A., Gervais, N., Fikry, C., Adamu Bukari, A.-R., Gerstein, A., Cuomo, C. A., Shapiro, R. S.. 2025-12-22. Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds. https://doi.org/10.64898/2025.12.19.695556

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