Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Feb;8(2):159-64.
doi: 10.1038/nmeth.1550. Epub 2011 Jan 9.

Knocking Out Multigene Redundancies via Cycles of Sexual Assortment and Fluorescence Selection

Free PMC article

Knocking Out Multigene Redundancies via Cycles of Sexual Assortment and Fluorescence Selection

Yo Suzuki et al. Nat Methods. .
Free PMC article


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.


Figure 1
Figure 1
Design of the Green Monster process. (a) Schematic overview of the process In yeast, crossing different haploid single-mutants generates 0-deletion (off-white), 1-deletion (light green), and 2-deletion (dark green) cells. From this mixture, flow cytometry enriches for 2-deletion cells. Higher-order multi-mutants are assembled via repeated rounds of sexual assortment and enrichment. (b) Universal GFP deletion cassette replaces KanMX4 in 'your favorite gene' (YFG) via recombination within TEF subsequences, while maintaining barcodes (stripes) intact. The inducible tetO2 promoter allows titration of GFP expression. Transcriptional terminators (brown) and the TEF promoter (light blue) and terminator (dark blue) are shown. Selectable marker: URA3.. (c) GMToolkits, inserted at the CAN1 locus, contain rtTA, either KanMX4 and STE2pr-Sp-his5 (GMToolkit-a) or NatMX4 and STE3pr-LEU2 (GMToolkit-α).
Figure 2
Figure 2
Demonstration of the Green Monster process. (a) Simulations showing that >99% of a cell population accumulate all 24 deletions in eight (top), 12 (middle), or 19 rounds (bottom), with greater efficiency for lower coefficient of variation (CV) of GFP intensity (achievable using internal standard to control for noise (b) Simulation showing that 24 linked deletions with the meiotic crossover probability between adjacent loci (P(R)) of 5% can assemble in 16 rounds when the GFP CV is 50%. (c) Cell sorting strategy. Histograms (y-axis: the percentage of maximum cell number) are shown for GFP intensity in no-GFP cells (0Δ), single-GFP cells (1Δ), and a haploid ‘meiotic mix’ resulting from a cross of two single-GFP strains, with an expected 1:2:1 ratio of no-GFP, one-GFP, and two-GFP cells. The brightest 1% of the cells in the meiotic mix were collected (red filled area). (d) GFP intensity of multi-mutants. Histograms are shown for no-GFP, 1-GFP, 2-GFP, 4-GFP, 8-GFP and 16-GFP ‘ABC16-monster’ cells (isogenic populations). (e) Fluorescence micrographs showing non-mutant (top left), double-mutant (top right), the ABC16-monster (bottom left), and a mixture of double mutant and ABC16-monster (bottom right) cells. Identical exposure, brightness, and contrast settings were used for images. Scale bar indicates 10 µm. (f) The en masse Green Monster process. Average deletion numbers for each round from three independent processes are plotted (red, blue, and yellow). Error bars indicate standard deviations (n = 21 – 34).
Figure 3
Figure 3
Hypersensitivity of the ABC16-monster to drugs. (a) Pie chart showing the number of drugs to which the ABC16-monster or the previously-described drug-hypersensitive AD strain is more sensitive. (b) A comparison of ABC16-monster and single-mutant drug sensitivity. The IC50 for each strain/drug combination relative to that of the corresponding non-mutant/drug combination was calculated. Area of each shape is proportional to this relative IC50 value. *: The minimum value among the relative IC50 values for single mutants is indicated for comparison with the relative IC50 of the ABC16-monster (c) Exponential growth rates of the ABC16-monster (green), non-mutant (blue), and single-deletion strains (grey) as a function of concentration of tamoxifen, fluconazole, and valinomycin.

Similar articles

See all similar articles

Cited by 30 articles

See all "Cited by" articles


    1. Tong AH, et al. Global mapping of the yeast genetic interaction network. Science. 2004;303:808–813. - PubMed
    1. Costanzo M, et al. The genetic landscape of a cell. Science. 2010;327:425–431. - PMC - PubMed
    1. Beh CT, Cool L, Phillips J, Rine J. Overlapping functions of the yeast oxysterol-binding protein homologues. Genetics. 2001;157:1117–1140. - PMC - PubMed
    1. Pruess M, et al. The Proteome Analysis database: a tool for the in silico analysis of whole proteomes. Nucleic Acids Res. 2003;31:414–417. - PMC - PubMed
    1. Decottignies A, et al. ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J. Biol. Chem. 1998;273:12612–12622. - PubMed

Publication types

MeSH terms


LinkOut - more resources