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.
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This work was supported by an NSERC Discovery Grant, an NSERC Discovery Accelerator Supplement, and a Banting Research Foundation Discovery Award to R.S.S. A.C. was supported by the Burroughs Wellcome Fund Career Award for Medical Scientists.
The authors declare no competing interests.
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Key reference using this protocol
Shapiro, R. S. et al. Nat. Microbiol. 3, 73–82 (2018): https://doi.org/10.1038/s41564-017-0043-0
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Halder, V., Porter, C.B.M., Chavez, A. et al. Design, execution, and analysis of CRISPR–Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans. Nat Protoc 14, 955–975 (2019). https://doi.org/10.1038/s41596-018-0122-6
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