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

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.

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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.

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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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Supplementary Information

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

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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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