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Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection

Nature Methods volume 8, pages 159–164 (2011)Cite this article

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Abstract

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

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Figure 1: Design of the green monster process.
Figure 2: Demonstration of the green monster process.
Figure 3: Hypersensitivity of the ABC16 monster to drugs.

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Acknowledgements

This work was supported by US National Institutes of Health grants R01 HG003224 and R21 CA130266 to F.P.R. O.D.K. was supported by a National Research Service Award fellowship from the National Institutes of Health–National Human Genome Research Institute. Micrographs were generated at Nikon Imaging Center at Harvard Medical School. We are grateful for yeast strains from C. Boone (University of Toronto) and A. Goffeau (Université catholique de Louvain), and for advice and assistance from M. Al-Shawi, B.J. Andrews, C. Baisden, R. Balasubramanian, W. Bender, C. Boone, R. Brost, S. Buratowski, D. Chowdhury, G.M. Church, D. Coen, E. Craig, E. Elion, M. Fenerjian, D. Gibson, J. Glass, A. Goffeau, R. Grene, C. Hutchison, S. Iwase, M. Johnston, B. Karas, K. Kono, K. Kuchler, J. Li, D. Morgan, S. Moye-Rowley, J. Murai, S. Oliver, F. Ozbek, Y. Pan, D. Pellman, A. Ramon, J. Rine, A. Rowat, P. Silver, H. Smith, M. Springer, K. Struhl, C. Tagwerker, M. Takahashi, B. Turcotte, D. Weitz and S. Yoshida; members of the Roth Lab, especially M. Tasan, J. Mellor, J. Komisarof, J. MacKay, A. Derti and S. Komili; and members of the Dana-Farber Center for Cancer Systems Biology, especially P. Braun, A. Dricot, D. Hill, Q. Li and H. Yu.

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Author notes

  1. Yo Suzuki, Oliver D King & Frederick P Roth

    Present address: Present addresses: Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, San Diego, California, USA (Y.S.), Boston Biomedical Research Institute, Watertown, Massachusetts, USA (O.D.K.) and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto and Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada (F.P.R.).,

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Yo Suzuki, Ramamurthy Mani, Oliver D King, Weidong Chen, Linda Pham, Lan V Zhang & Frederick P Roth

  2. Stanford Genome Technology Center, Palo Alto, California, USA.,

    Robert P St Onge

  3. Bioinformatics Program, Boston University, Boston, Massachusetts, USA

    Adrian Heilbut & Joseph Lehár

  4. Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Vyacheslav M Labunskyy & Vadim N Gladyshev

  5. Genome Research Centre, The University of Hong Kong, Pokfulam, Hong Kong, China

    Amy H Y Tong

  6. Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Corey Nislow

  7. Department of Pharmaceutical Sciences, Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Guri Giaever

  8. Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Marc Vidal & Frederick P Roth

  9. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Marc Vidal

  10. Flow Cytometry Core Facility, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Peter Schow

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Contributions

Y.S. and F.P.R. developed the green monster method and prepared the manuscript; R.P.S.O., A.H., J.L. and Y.S. measured drug sensitivity; R.M. analyzed growth curves; O.D.K. simulated the process; L.V.Z., C.N. and G.G. advised on method design; A.H.Y.T., V.M.L., V.N.G. and M.V. provided reagents and advice; W.C. and L.P. provided technical support; P.S. and Y.S. performed flow cytometry.

Corresponding authors

Correspondence to Yo Suzuki or Frederick P Roth.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8 and Supplementary Tables 1–13 (PDF 4210 kb)

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Suzuki, Y., Onge, R., Mani, R. et al. Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection. Nat Methods 8, 159–164 (2011). https://doi.org/10.1038/nmeth.1550

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