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One-step targeted gene deletion in Candida albicans haploids

Abstract

The recent discovery of haploids in Candida albicans and the construction of tool strains carrying multiple auxotrophic markers have enabled, for the first time, performing one-step gene deletions in this fungal human pathogen. This breakthrough promises to greatly facilitate the molecular and genetic study of C. albicans biology and pathogenicity. However, the construction of gene-deletion mutants in C. albicans haploids involves many technical difficulties, particularly low transformation efficiency and autodiploidization. Here we describe a highly effective protocol for designing and performing one-step gene deletion in C. albicans haploids, which takes 11 d to complete (not including plasmid construction, which may take 2 weeks). A gene deletion cassette is constructed on a plasmid and subsequently released for transformation by lithium acetate incubation or electroporation. Desired gene-deletion mutants are identified and their ploidy is assessed simultaneously by colony PCR before final confirmation by flow cytometry.

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Figure 1
Figure 2: Schematic diagram of the gene deletion construct.
Figure 3: Visualization of the gene deletion cassette.
Figure 4: Examples of transformants on plates.
Figure 5: Characterization of transformants by colony PCR.
Figure 6: Ploidy analysis of transformants by flow cytometry.

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Acknowledgements

We thank J. Berman and members of the Wang lab for critical reading of the manuscript. This work was funded by the Agency for Sciences, Technology and Research of Singapore.

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Contributions

G.Z. designed and performed the experiments and wrote the first draft of the manuscript. F.Y.C. contributed to plasmid construction, and Y.-M.W. helped with flow cytometry analysis. Y.W. discussed and commented on the results at all stages and revised the manuscript.

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Correspondence to Yue Wang.

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The authors declare no competing financial interests.

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Zeng, G., Wang, YM., Chan, F. et al. One-step targeted gene deletion in Candida albicans haploids. Nat Protoc 9, 464–473 (2014). https://doi.org/10.1038/nprot.2014.029

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