A CRISPR Cas9-based gene drive platform for studying complex genetic interactions in Candida albicans

The opportunistic pathogen Candida albicans a the leading cause of fungal infection worldwide. Yet, genetic analysis in this clinically important pathogen remains cumbersome because it is a diploid organism that requires sequential allele knockouts to generate homozygous null mutants. This issue is further compounded when double mutant lines need to be generated, such as when studying critical genetic networks modulating virulence pathways. Here, we have developed a CRISPR-Cas9-based ‘gene drive’ platform for rapid, precise, and efficient genome editing in C. albicans, enabling applications for global genetic analysis of fungal pathogenesis. In our gene drive system, a modified DNA donor molecule is used that acts as a selfish genetic element, replaces the targeted site, and propagates to replace any additional wild-type locus it encounters. Coupling this approach with newly identified mating-competent haploid C. albicans lineages, we can rapidly and efficiently create diploid C. albicans strains that are double homozygous deletion mutants, enabling us to create large scale double-deletion libraries and analyze complex genetic interactions networks in C. albicans for the first time. We demonstrate the power of this technology by generating two double-gene deletion libraries, targeting factors involved in either drug efflux or cellular adhesion. By screening these libraries for sensitivity to antifungal perturbations or biofilm growth, we identify central regulators of these pathways, and determine how genetic interaction networks shift under diverse environmental conditions. This platform transforms our ability to perform complex genetic interaction analysis of virulence traits in C. albicans and could be readily extended to other clinically important fungal pathogens.