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A CRISPR–Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

Nature Microbiology volume 3, pages 73–82 (2018)Cite this article

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Abstract

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR–Cas9-based ‘gene drive array’ platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens.

Fungal pathogens are a leading cause of human mortality among the ever-growing immunocompromised population. The most pervasive cause of fungal infections is Candida albicans, an opportunistic pathogen present in the human microbiome1. As with other fungal infections, treating C. albicans is challenging due to its close evolutionary relationship with its human host. Indeed, identifying antifungal agents without toxicity to humans has proved incredibly challenging—there are few classes of antifungals in clinical use and the emergence of antifungal resistance is common, resulting in limited treatment options for these deadly infections1. In addition, C. albicans readily forms robust biofilms on medical devices—including urinary and venous catheters—that are highly resistant to antifungal treatment2 and result in the highest crude mortality rate for medical-device-associated infections3.

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Fig. 1: A synthetic Cas9 gene drive system for targeted homozygous deletion in C. albicans.
Fig. 2: Optimized C. albicans gene drive efficiently creates homozygous double-gene deletion strains.
Fig. 3: Fitness and genetic interaction analysis of two double-gene deletion virulence libraries.
Fig. 4: Double-deletion matrix of C. albicans transporter genes reveals condition-specific sensitivity and genetic interactions.
Fig. 5: C. albicans double adhesin deletion matrix highlights critical factors and genetic interactions for biofilm growth.

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Acknowledgements

We thank G. Fink, J. Berman, M. Hickman, V. Vyas and A. Baryshnikova for helpful discussions. We also thank V. Vyas, J. Köhler and L. Cowen for strains. This work was supported by the Paul G. Allen Frontiers Group, a Banting postdoctoral fellowship from the Canadian Institutes of Health Research, National Cancer Institute grantno. 5T32CA009216-34, US National Institutes of Health National Human Genome Research Institute grant no. RM1 HG008525 and the Wyss Institute for Biologically Inspired Engineering.

Author information

Author notes

  1. Alejandro Chavez

    Present address: Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, 10032, NY, USA

  2. Rebecca S. Shapiro and Alejandro Chavez contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Engineering, Institute for Medical Engineering and Science, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Rebecca S. Shapiro, Caroline B. M. Porter & James J. Collins

  2. Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA

    Rebecca S. Shapiro, Caroline B. M. Porter, Meagan Hamblin & James J. Collins

  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA

    Rebecca S. Shapiro, Alejandro Chavez, Christian S. Kaas, George M. Church & James J. Collins

  4. Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA

    Alejandro Chavez

  5. Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA

    Alejandro Chavez, Christian S. Kaas, James E. DiCarlo & George M. Church

  6. Department of Expression Technologies 2, Novo Nordisk A/S, Maaloev, 2760, Denmark

    Christian S. Kaas

  7. Department of Ophthalmology, Columbia University, New York, NY, 10032, USA

    James E. DiCarlo

  8. Institute of Molecular and Cell Biology, Agency for Science, Technology & Research, 61 Biopolis Drive (Proteos), Singapore, 138673, Singapore

    Guisheng Zeng, Xiaoli Xu & Yue Wang

  9. Department of BioSciences, Rice University, Houston, TX, 77005, USA

    Alexey V. Revtovich & Natalia V. Kirienko

  10. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117549, Singapore

    Yue Wang

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Contributions

R.S.S., A.C., J.E.D., G.M.C. and J.J.C. conceptualized the project; R.S.S., A.C., M.H., A.V.R. and X.X. performed the experiments; C.B.M.P., C.S.K. and R.S.S. performed analysis and visualization of experimental results; G.Z. and Y.W. generated and provided strains; R.S.S., A.C. and C.B.M.P wrote and edited the manuscript; Y.W., N.V.K., G.M.C. and J.J.C. supervised the project; J.J.C and G.M.C. acquired funding.

Corresponding authors

Correspondence to George M. Church or James J. Collins.

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The authors declare no competing financial interests.

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Supplementary Information

Supplementary Figures 1–4, Supplementary Figure legends, Supplementary Table legends and Supplementary Notes.

Life Sciences Reporting Summary

Supplementary Table 1

Gene drive construct variants. Related to Fig. 2. This table summarizes the different gene drive construct variants used as part of the optimization of the C. albicans gene drive system.Gene drive construct variants. Related to Fig. 2. This table summarizes the different gene drive construct variants used as part of the optimization of the C. albicans gene drive system.

Supplementary Table 2

C. albicans efflux and adhesin genes targeted for deletion, and library matrix summary. Related to Fig. 3. This table summarizes the different C. albicans adhesin and efflux genes targeted for deletion, and lists each single- and double-gene deletion strains generated as part of this study.C. albicans efflux and adhesin genes targeted for deletion, and library matrix summary. Related to Fig. 3. This table summarizes the different C. albicans adhesin and efflux genes targeted for deletion, and lists each single- and double-gene deletion strains generated as part of this study.

Supplementary Table 3

Whole-genome sequencing summary of gene drive deletion strains. Related to Figs. 2 and 3. This table summarizes the results of whole-genome sequencing, and lists each gene found to be deleted in different strain backgrounds, as well as sequence coverage information.

Supplementary Table 4

Genetic interaction scores and significant genetic interactions for double-gene deletion libraries. Related to Figs. 2–4. This table lists genetic interactions scores (calculated using a multiplicative model) and significant positive and negative genetic interactions for both C. albicans double-gene deletion libraries (efflux and adhesin mutants).

Supplementary Table 5

Summary of antifungal perturbations for drug efflux pump deletion screening. Related to Fig. 4. This table lists all perturbation conditions used for screening the C. albicans efflux pump library, including the concentration of drug tested in the screen.

Supplementary Text File 1

Table of gene drive construct variants and optimization.

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Shapiro, R.S., Chavez, A., Porter, C.B.M. et al. A CRISPR–Cas9-based gene drive platform for genetic interaction analysis in Candida albicans . Nat Microbiol 3, 73–82 (2018). https://doi.org/10.1038/s41564-017-0043-0

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