LAUSR

The study of fungal pathogens is of immediate importance, yet progress is hindered by the technical challenges of genetic manipulation. For Candida species, their inability to maintain plasmids, unusual codon usage, and inefficient homologous recombination are among the obstacles limiting efficient genetic manipulation. New advances in genomic biotechnologies—particularly CRISPR-based tools—have revolutionized genome editing for many fungal species. Here, we present a protocol for CRISPR–Cas9-based manipulation in Candida albicans using a modified gene-drive-based strategy that takes ~1 month to complete. We detail the generation of Candida-optimized Cas9-based plasmids for gene deletion, an efficient transformation protocol using C. albicans haploids, and an optimized mating strategy to generate homozygous single- and double-gene diploid mutants. We further describe protocols for quantifying cell growth and analysis pipelines to calculate fitness and genetic interaction scores for genetic mutants. This protocol overcomes previous limitations associated with genetic manipulation in C. albicans and advances researchers’ ability to perform genetic analysis in this pathogen; the protocol also has broad applicability to other mating-competent microorganisms.Here, the authors provide protocols for CRISPR–Cas9-based genetic manipulation of Candida albicans, using a gene-drive strategy that allows genetic interaction networks to be established for this important fungal pathogen.