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Genome-wide C-SWAT library for high-throughput yeast genome tagging

Nature Methods volume 15, pages 598–600 (2018)Cite this article

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Abstract

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.

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Fig. 1: Design, construction, and validation of the C-SWAT library.
Fig. 2: High-throughput protein tagging with the C-SWAT library.

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Acknowledgements

We thank the CellNetworks Deep Sequencing Core Facility (Heidelberg University) and the Genomics Core Facility (EMBL), and acknowledge generous support from the DFG for data storage (LDSF2). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center SFB1036 (M.K., A.K., T.P.D., and M.K.L.), the Weizmann Institute of Science (E.S. and E.D.L.), the China Scholarship Council (Y.D.), fellowships from the HBIGS graduate school (I.K., K.H., and F.H.), an SFB1036 travel grant (E.S.), and the Alexander von Humboldt Foundation (M.Š.), and partially by DFG grant KN498/11-1 (M.K.), Israel Science Foundation grants 1775/12 and 2179/14 (E.D.L.), and HFSP Career Development Award CDA00077/2015 (E.D.L.).

Author information

Author notes

  1. Florian Huber

    Present address: Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

  2. Martin Štefl

    Present address: Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry of the CAS, Prague, Czech Republic

  3. Anton Khmelinskii

    Present address: Institute of Molecular Biology (IMB), Mainz, Germany

  4. These authors contributed equally: Matthias Meurer, Yuanqiang Duan, Ehud Sass.

Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

    Matthias Meurer, Yuanqiang Duan, Ilia Kats, Konrad Herbst, Benjamin C. Buchmuller, Verena Dederer, Florian Huber, Martin Štefl, Marius K. Lemberg, Anton Khmelinskii & Michael Knop

  2. Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

    Ehud Sass & Emmanuel D. Levy

  3. Cell Morphogenesis and Signal Transduction, DKFZ-ZMBH Alliance and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Daniel Kirrmaier & Michael Knop

  4. Division of Redox Regulation, DKFZ-ZMBH Alliance and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Koen Van Laer & Tobias P. Dick

Authors
  1. Matthias Meurer
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  2. Yuanqiang Duan
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  4. Ilia Kats
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Contributions

M.K., M.M., E.D.L., and A.K. planned the work. Y.D. and M.M., together with E.S., constructed the library, with help from B.C.B., V.D., K.H., F.H., D.K., I.K., M.Š., K.V.L., A.K., and M.K. E.S. and E.D.L. developed Anchor-Seq and E.D.L. analyzed the sequencing data, with input from M.M. and K.H. Y.D., M.M., I.K., A.K., and D.K. generated and analyzed the mNeonGreen and mScarlet-I libraries. A.K. and M.K. wrote the manuscript with E.D.L. and E.S., with input from all other authors.

Corresponding authors

Correspondence to Anton Khmelinskii, Emmanuel D. Levy or Michael Knop.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Anchor-Seq workflow.

(a) Genomic DNA is extracted from a pooled library, where a different ORF (magenta) is modified (e.g., tagged with the C-SWAT acceptor module (green)) in each strain. The DNA is sheared and the fragment ends are processed to allow A-T ligation of vectorette adaptors. Selective amplification of fragments containing the library tag is achieved with two primers: a tag primer (green) that anneals to the tag sequence and a vectorette primer (blue) that has the same sequence as the vectorette adaptor and thus cannot anneal to it. The sequence complementary to the vectorette primer is produced in the first PCR cycle initiated by the tag primer. Exponential amplification only of fragments containing the library tag can subsequently occur (15 PCR cycles in total). The PCR product is purified and subjected to another 15 PCR cycles to add the P5 and P7 Illumina sequences to the amplified fragments. (b) Sequence of the vectorette primer (blue) in relation to the central portion of the vectorette adaptor and the product of the priming cycle with the tag primer (selective amplification step in (a)).

Supplementary Figure 2 Efficiency of tag swapping with different donors.

(a) Three types of donor plasmids for tag swapping with C-SWAT strains. The type I donor is used for markerless tagging. In this donor the tag is placed between short homology arms (gray) and flanked by I-SceI cut sites (black squares). Tag swapping events are selected by resistance to 5-fluoroorotic acid (5-FOA), which indicates loss of the URA3 selection marker. The type II donor is used for tagging with reconstitution of the hph selection marker. In this donor the left homology arm (light gray) is followed by the tag, the terminator sequence of the ADH1 gene from Saccharomyces cerevisiae, the promoter sequence of the TEF gene from Ashbya gossypii and the hphΔC sequence coding for an N-terminal fragment (amino acids 1-192) of the hph (hygromycin resistance-encoding gene) marker. Tag swapping events are selected by resistance to 5-FOA and later/alternatively hygromycin, which indicates reconstitution of the hph selection marker. The type III donor is used for tagging with a new selection marker. In this donor the left homology arm (light gray) is followed by the tag, the ADH1 terminator sequence and a selection marker (e.g., the kanMX marker (resistance to G-418)). Tag swapping events are selected by resistance to 5-FOA and later G-418. (b) Comparison of tagging efficiency using three donors with the mNeonGreen fluorescent protein. Protein tagging was performed with 20 C-SWAT strains for the indicated genes. Full distributions of single-cell mNeonGreen fluorescence intensities measured with flow cytometry (~20000 cells per strain). The percentage of cells with fluorescence above background (fluorescence of a wild type strain, dashed line) is indicated in the plots.

Supplementary Figure 3 Brightness of different fluorescent proteins in yeast.

(a) Relative brightness of different green fluorescent proteins (greenFPs) in yeast. Each greenFP was fused to mCherry, with Don1 as a spacer protein to minimize Förster resonance energy transfer (FRET) between the two fluorescent proteins. The fusions were expressed in yeast from the strong constitutive GPD promoter. Whole colony greenFP fluorescence intensities, measured with two sets of excitation and emission wavelengths, were normalized for protein expression levels using mCherry fluorescence intensities. The resulting relative brightness estimates were normalized to sfGFP (mean ± s.d., n = 5 biological replicates each with 4 technical replicates). Based on these results and excitation/emission spectra, we estimate that mNeonGreen with 505/516 nm excitation/emission is ~2 fold brighter than sfGFP with 488/510 nm excitation/emission. (b) Relative brightness of different red fluorescent proteins (redFPs) in yeast. Each redFP was fused to sfGFP with Don1 as a spacer protein. The fusions were expressed from the GPD promoter and whole colony redFP fluorescence intensities, measured with two sets of excitation and emission wavelengths, were normalized for protein expression levels using sfGFP fluorescence intensities. The resulting relative brightness estimates were normalized to mCherry (mean ± s.d., n = 3 biological replicates each with 4 technical replicates). Based on these results and excitation/emission spectra, we estimate that mScarlet-I with 569/593 nm excitation/emission is ~3 fold brighter than mCherry with 587/610 nm excitation/emission.

Supplementary Figure 4 Measurements of protein abundance with mNeonGreen and mScarlet-I libraries.

Fluorescence measurements of colonies in three technical replicates for the mNeonGreen and mScarlet-I libraries and negative control strains not expressing a fluorescent protein. (a) Distribution of fluorescence intensities of negative control colonies used to correct colony fluorescence measurements in mNG-I, mNG-II and mSC-II libraries for background. Less than 0.5% of negative control colonies (33 and 72 out of 15597 for mNeonGreen and mScarlet-I channels, respectively) exceeded the threshold set at 1.2 (red dashed lines), above which a tagged protein was considered to be expressed at detectable levels. (b) Comparison of relative protein expression levels measured with the mNG-I, mNG-II and mSC-II libraries. The libraries were derived from the C-SWAT library by tag swapping with the three donor plasmids in Fig. 2a. Fluorescence intensities of colonies were corrected for background fluorescence. (c) Comparison between relative protein expression levels measured with the mNG-I library and estimates of absolute protein abundance from ref. 1 (below). Absolute estimates are based on measurements of protein abundance with mass spectrometry of wild type yeast2, fluorescence microscopy and flow cytometry of strains expressing GFP fusions3,4 or immunoblotting of TAP-tagged proteins5. Only strains with fluorescence intensities at least 1.2 fold above background were used in (b) and (c). ρ, Spearman’s rank correlation coefficient.

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Supplementary Figure 5 Localization and expression of previously undetected proteins.

(a) Cartoon of a genomic locus with 4 overlapping ORFs (left panel). YPR170W-B and YPR169W-A have the same start codon but the coding sequence of YPR170W-B is interrupted by an intron. Fluorescence microscopy of the corresponding strains from the mNG-I library (right panel). All images were acquired identically as 16-bit intensity images but are shown with different lower and upper limits of the display range: 100-10000 for Ypr170w-b and Ypr169w-a, 100-600 for Ypr170w-a and Ypr170c. Note that Ypr170w-a localizes to vacuoles (Fig. 2f) but its expression is extremely low compared to Ypr170w-b and Ypr169w-a. A representative section from one out of three fields of view is shown for each strain. Scale bar, 5 µm. (b) Fluorescence levels of mNG-I strains for two groups of ORFs: annotated as dubious as of January 2018 (dubious) and reclassified from dubious to verified or uncharacterized between July 2016 and January 2018 (reclassified) (Supplementary Table 4). Fluorescence intensities of colonies are expressed in units of background fluorescence, not corrected for background fluorescence.

Supplementary information

Supplementary Text and Figure

Supplementary Figs. 1–5, Supplementary Tables 5–7, and Supplementary Notes 1 and 2

Reporting Summary

Supplementary Table 1

C-SWAT library description

Supplementary Table 2

Protein expression levels in mNG-I, mNG-II, and mSC-II libraries

Supplementary Table 3

Expression and localization of previously undetected proteins

Supplementary Table 4

Changes in classification of dubious ORFs

Supplementary Data

Montages of fluorescence microscopy images for 32 previously undetected proteins

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Meurer, M., Duan, Y., Sass, E. et al. Genome-wide C-SWAT library for high-throughput yeast genome tagging. Nat Methods 15, 598–600 (2018). https://doi.org/10.1038/s41592-018-0045-8

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