Brief Communication | Published:

High-throughput, image-based screening of pooled genetic-variant libraries

Nature Methods volume 14, pages 11591162 (2017) | Download Citation

Abstract

We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in individual cells. We achieve genotyping by introducing barcoded genetic variants into cells as pooled libraries and reading the barcodes out using massively multiplexed fluorescence in situ hybridization. To demonstrate the power of image-based pooled screening, we identified brighter and more photostable variants of the fluorescent protein YFAST among 60,000 variants.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Protein Data Bank

References

  1. 1.

    & PCR Methods Appl. 2, 28–33 (1992).

  2. 2.

    & Nat. Methods 11, 499–507 (2014).

  3. 3.

    , , & Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

  4. 4.

    et al. Nature 478, 204–208 (2011).

  5. 5.

    et al. Nat. Biotechnol. 29, 942–947 (2011).

  6. 6.

    et al. Nat. Methods 5, 545–551 (2008).

  7. 7.

    , , , & Lab Chip 13, 2320–2327 (2013).

  8. 8.

    et al. Integr. Biol. 7, 263–273 (2015).

  9. 9.

    , & Nat. Rev. Genet. 16, 299–311 (2015).

  10. 10.

    et al. Cell 167, 1853–1866.e17 (2016).

  11. 11.

    et al. Cell 167, 1867–1882.e21 (2016).

  12. 12.

    et al. Cell 167, 1883–1896.e15 (2016).

  13. 13.

    et al. Nat. Methods 14, 297–301 (2017).

  14. 14.

    Proc. Natl. Acad. Sci. USA 113, 497–502 (2016).

  15. 15.

    , , , & Science 348, aaa6090 (2015).

  16. 16.

    et al. Proc. Natl. Acad. Sci. USA 113, 11046–11051 (2016).

  17. 17.

    , , & Science 280, 585–590 (1998).

  18. 18.

    , , , & Nat. Methods 5, 877–879 (2008).

  19. 19.

    , , & PLoS One 6, e28674 (2011).

  20. 20.

    , , , & Proc. Natl. Acad. Sci. USA 111, 8452–8457 (2014).

  21. 21.

    , , , & eLife 3, e01775 (2014).

  22. 22.

    et al. Nat. Methods 6, 343–345 (2009).

  23. 23.

    & Nucleic Acids Res. 25, 1203–1210 (1997).

  24. 24.

    et al. Nat. Methods 9, 72–74 (2011).

  25. 25.

    , , & Proc. Natl. Acad. Sci. USA 109, 1347–1352 (2012).

  26. 26.

    , & Nat. Struct. Biol. 10, 663–668 (2003).

  27. 27.

    , , & PLoS One 8, e75120 (2013).

  28. 28.

    , & Nat. Methods 3, 891–893 (2006).

  29. 29.

    , & Protocol Exchange (2017).

Download references

Acknowledgements

We thank H. Babcock for instrumentation advice and many readers for participating in the discussion of the preprint of this paper that we deposited on bioRxiv. This work was supported in part by the NIH. X.Z. is an HHMI investigator.

Author information

Affiliations

  1. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

    • George Emanuel
    • , Jeffrey R Moffitt
    •  & Xiaowei Zhuang
  2. Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA.

    • George Emanuel
  3. Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

    • George Emanuel
    • , Jeffrey R Moffitt
    •  & Xiaowei Zhuang

Authors

  1. Search for George Emanuel in:

  2. Search for Jeffrey R Moffitt in:

  3. Search for Xiaowei Zhuang in:

Contributions

G.E., J.R.M., and X.Z. conceived the study and designed the experiments. G.E. performed experiments and analyzed data. G.E., J.R.M., and X.Z. interpreted the data and wrote the manuscript.

Competing interests

G.E., J.R.M., and X.Z. are inventors on a patent applied for by Harvard University that covers the screening method described here.

Corresponding authors

Correspondence to Jeffrey R Moffitt or Xiaowei Zhuang.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3, Supplementary Tables 1–2 and Supplementary Note 1

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    Library construction and imaging for high-throughput image-based screening using multiplexed FISH.

Zip files

  1. 1.

    Supplementary Software

    Software for analysis of image data and sequencing data.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.4495

Further reading