Protocol | Published:

Design, execution, and analysis of CRISPR–Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans

Nature Protocolsvolume 14pages955975 (2019) | Download Citation

Abstract

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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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

Change history

  • 11 April 2019

    The version of this paper originally published contained reference errors. The sentence “To dissect complex genetic interactions in C. albicans, a CRISPR–Cas9-based Gene Drive Array (GDA) was developed” incorrectly cited ref. 13, and should have cited ref. 14. In addition, the reference included as ref. 13 in the original paper was incorrect, and should have been the following: Shapiro, R. S., Chavez, A. & Collins, J. J. CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms. Nat. Rev. Microbiol. 16, 333–339 (2018). This reference should have been cited after the sentence “Recent innovations in CRISPR–Cas9-based genome editing have facilitated such genetic interaction analyses.” The original reference 13 (Gerami-Nejad, M., Zacchi, L. F., McClellan, M., Matter, K. & Berman, J. Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans. Microbiology 159, 565–579 (2013)) should have been cited later in the paper, and is now in the reference list as ref. 27. As a result, original references 27–33 have been renumbered in the reference list and in the text. These changes have been made in the PDF and HTML versions of the protocol.

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Acknowledgements

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.

Author information

Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

    • Viola Halder
    •  & Rebecca S. Shapiro
  2. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Caroline B. M. Porter
  3. Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, USA

    • Alejandro Chavez

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  1. Search for Viola Halder in:

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Contributions

The protocol was conceived and developed by C.B.M.P., A.C., and R.S.S. The manuscript was written by V.H. and R.S.S., with contributions from C.B.M.P. and A.C. Experiments were performed by V.H. All authors contributed to editing the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Rebecca S. Shapiro.

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DOI

https://doi.org/10.1038/s41596-018-0122-6

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