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

Nature Protocols volume 14, pages 955–975 (2019)Cite this article

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An Author Correction to this article was published on 11 April 2019

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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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Fig. 1: CRISPR–Cas9-based gene drive system to generate targeted homozygous single- and double-gene deletions in C. albicans.
Fig. 2: Primers designed for PCR diagnostic tests.
Fig. 3: Anticipated results of white-opaque switching and haploid matings.

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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.

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Authors and 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

Authors
  1. Viola Halder
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  2. Caroline B. M. Porter
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  3. Alejandro Chavez
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  4. Rebecca S. Shapiro
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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.

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Correspondence to Rebecca S. Shapiro.

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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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