A CRISPR–Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR–Cas9-based ‘gene drive array’ platform to facilitate efficient genetic analysis in C. albicans . In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens. A CRISPR–Cas9-based gene drive array platform is developed and combined with mating-competent Candida albicans haploids to generate homozygous double-deletion mutants, transforming our ability to do genetic interaction analyses in fungi.