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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Shapiro, R. S., Robbins, N. & Cowen, L. E. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol. Mol. Biol. Rev. 75, 213–267 (2011).

  2. 2.

    Nobile, C. J. & Johnson, A. D. Candida albicans biofilms and human disease. Annu. Rev. Microbiol. 69, 71–92 (2015).

  3. 3.

    Ramage, G., Mowat, E., Jones, B., Williams, C. & Lopez-Ribot, J. Our current understanding of fungal biofilms. Crit. Rev. Microbiol. 35, 340–355 (2009).

  4. 4.

    Tong, A. H. Y. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004).

  5. 5.

    Enkler, L., Richer, D., Marchand, A. L., Ferrandon, D. & Jossinet, F. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system. Sci. Rep. 6, 35766 (2016).

  6. 6.

    Fuller, K. K., Chen, S., Loros, J. J. & Dunlap, J. C. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot. Cell 14, 1073–1080 (2015).

  7. 7.

    Vyas, V. K., Barrasa, M. I. & Fink, G. R. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci. Adv. 1, e1500248 (2015).

  8. 8.

    Min, K., Ichikawa, Y., Woolford, C. A. & Mitchell, A. P. Candida albicans gene deletion with a transient CRISPR-Cas9 system. mSphere 1, e00130-16 (2016).

  9. 9.

    Nguyen, N., Quail, M. M. F. & Hernday, A. D. An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans. mSphere 2, e00149-17 (2017).

  10. 10.

    DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M. & Church, G. M. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat. Biotechnol. 33, 1250–1255 (2015).

  11. 11.

    Hickman, M. A. et al. The ‘obligate diploid’ Candida albicans forms mating-competent haploids. Nature 494, 55–59 (2013).

  12. 12.

    Miller, M. G. & Johnson, A. D. White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating. Cell 110, 293–302 (2002).

  13. 13.

    Cannon, R. D. et al. Efflux-mediated antifungal drug resistance. Clin. Microbiol. Rev. 22, 291–321 (2009).

  14. 14.

    Sundstrom, P. Adhesins in Candida albicans. Curr. Opin. Microbiol. 2, 353–357 (1999).

  15. 15.

    Baryshnikova, A., Costanzo, M., Myers, C. L., Andrews, B. & Boone, C. Genetic interaction networks: toward an understanding of heritability. Annu. Rev. Genomics Hum. Genet. 14, 111–133 (2013).

  16. 16.

    Boone, C., Bussey, H. & Andrews, B. J. Exploring genetic interactions and networks with yeast. Nat. Rev. Genet. 8, 437–449 (2007).

  17. 17.

    Mani, R., St Onge, R. P., Hartman, J. L. 4th, Giaever, G. & Roth, F. P. Defining genetic interaction. Proc. Natl Acad. Sci. USA 105, 3461–3466 (2008).

  18. 18.

    Morschhäuser, J. The genetic basis of fluconazole resistance development in Candida albicans. Biochim. Biophys. Acta 1587, 240–248 (2002).

  19. 19.

    Hawser, S. P. & Douglas, L. J. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect. Immun. 62, 915–921 (1994).

  20. 20.

    Lawrence, E. L. & Turner, I. G. Materials for urinary catheters: a review of their history and development in the UK. Med. Eng. Phys. 27, 443–453 (2005).

  21. 21.

    Fuller, KevinK. J. C. R. Protein kinase A and fungal virulence:a sinister side to a conserved nutrient sensing pathway. Virulence 3, 109 (2012).

  22. 22.

    Sibley, L. D., Howlett, B. J. Heitman, J. (eds) Evolution of Virulence in Eukaryotic Microbes (Wiley, USA, 2012).

  23. 23.

    Shapiro, R. S., Ryan, O., Boone, C. & Cowen, L. E. Regulatory circuitry governing morphogenesis in Saccharomyces cerevisiae and Candida albicans. Cell Cycle 11, 4294–4295 (2012).

  24. 24.

    Cui, Z., Hirata, D., Tsuchiya, E., Osada, H. & Miyakawa, T. The multidrug resistance-associated protein (MRP) subfamily (Yrs1/Yor1) of Saccharomyces cerevisiae is important for the tolerance to a broad range of organic anions. J. Biol. Chem. 271, 14712–14716 (1996).

  25. 25.

    Tomitori, H., Kashiwagi, K., Sakata, K., Kakinuma, Y. & Igarashi, K. Identification of a gene for a polyamine transport protein in yeast. J. Biol. Chem. 274, 3265–3267 (1999).

  26. 26.

    Costanzo, M. et al. The genetic landscape of a cell. Science 327, 425–431 (2010).

  27. 27.

    Byrne, K. P. & Wolfe, K. H. The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res. 15, 1456–1461 (2005).

  28. 28.

    Vallabhaneni, S. et al. Investigation of the first seven reported cases of Candida auris, a globally emerging invasive, multidrug-resistant fungus-United States, May 2013-August 2016. Am. J. Transplant 17, 296–299 (2017).

  29. 29.

    Chatterjee, S. et al. Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics 16, 686 (2015).

  30. 30.

    Nobile, C. J. et al. Complementary adhesin function in C. albicans biofilm formation. Curr. Biol. 18, 1017–1024 (2008).

  31. 31.

    Liu, Y. & Filler, S. G. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryot. Cell 10, 168–173 (2011).

  32. 32.

    Shapiro, R. S., Zaas, A. K., Betancourt-Quiroz, M., Perfect, J. R. & Cowen, L. E. The Hsp90 co-chaperone Sgt1 governs Candida albicans morphogenesis and drug resistance. PLoS ONE 7, e44734 (2012).

  33. 33.

    Shen, J., Guo, W. & Köhler, J. R. CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species. Infect. Immun. 73, 1239–1242 (2005).

  34. 34.

    Shapiro, R. S. et al. Pho85, Pcl1, and Hms1 signaling governs Candida albicans morphogenesis induced by high temperature or Hsp90 compromise. Curr. Biol. 22, 461–470 (2012).

  35. 35.

    Ryan, O. et al. Global gene deletion analysis exploring yeast filamentous growth. Science 337, 1353–1356 (2012).

  36. 36.

    Ramage, G., Vande Walle, K., Wickes, B. L. & López-Ribot, J. L. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother. 45, 2475–2479 (2001).

  37. 37.

    Robbins, N. et al. Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog. 7, e1002257 (2011).

  38. 38.

    St Onge, R. P. et al. Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions. Nat. Genet. 39, 199–206 (2007).

  39. 39.

    Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

  40. 40.

    Warnes, G. R. et al. gplots. R Package v.3.0.1 (CRAN, 2016); https://CRAN.R-project.org/package=gplots

  41. 41.

    RStudio (accessed 6 June 2017); https://www.rstudio.com

  42. 42.

    R: The R Project for Statistical Computing (accessed 6 June 2017); https://www.R-project.org

  43. 43.

    Baym, M. et al. Inexpensive multiplexed library preparation for megabase-sized genomes. PLoS ONE 10, e0128036 (2015).

  44. 44.

    Cohen, N. R. et al. A role for the bacterial GATC methylome in antibiotic stress survival. Nat. Genet. 48, 581–586 (2016).

  45. 45.

    Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).

  46. 46.

    Kaas, C. S., Kristensen, C., Betenbaugh, M. J. & Andersen, M. R. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy. BMC Genomics 16, 160 (2015).

  47. 47.

    Ihaka, R. & Gentleman, R. R: A language for data analysis and graphics. J. Comput. Graph. Stat. 5, 299 (1996).

Download references


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

    • Alejandro Chavez

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

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


  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


  1. Search for Rebecca S. Shapiro in:

  2. Search for Alejandro Chavez in:

  3. Search for Caroline B. M. Porter in:

  4. Search for Meagan Hamblin in:

  5. Search for Christian S. Kaas in:

  6. Search for James E. DiCarlo in:

  7. Search for Guisheng Zeng in:

  8. Search for Xiaoli Xu in:

  9. Search for Alexey V. Revtovich in:

  10. Search for Natalia V. Kirienko in:

  11. Search for Yue Wang in:

  12. Search for George M. Church in:

  13. Search for James J. Collins in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Electronic supplementary material

  1. Supplementary Information

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

  2. Life Sciences Reporting Summary

  3. 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.

  4. 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.

  5. 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.

  6. 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).

  7. 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.

  8. Supplementary Text File 1

    Table of gene drive construct variants and optimization.

About this article

Publication history






Further reading