Brief Communication | Published:

Genetic interaction mapping in mammalian cells using CRISPR interference

Nature Methods volume 14, pages 577580 (2017) | Download Citation

Abstract

We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein–protein-interaction data revealed a functional map of chromatin regulation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Cell 141, 739–745 (2010).

  2. 2.

    et al. Nature 446, 806–810 (2007).

  3. 3.

    & Nat. Biotechnol. 23, 561–566 (2005).

  4. 4.

    et al. Science 322, 405–410 (2008).

  5. 5.

    et al. Science 353, aaf1420 (2016).

  6. 6.

    et al. eLife 4, e05464 (2015).

  7. 7.

    et al. Nat. Methods 8, 341–346 (2011).

  8. 8.

    et al. Nat. Methods 10, 432–437 (2013).

  9. 9.

    , , , & Nat. Methods 10, 427–431 (2013).

  10. 10.

    Nature 418, 244–251 (2002).

  11. 11.

    , , & Nat. Biotechnol. 34, 634–636 (2016).

  12. 12.

    et al. Cell 154, 442–451 (2013).

  13. 13.

    et al. Nat. Biotechnol. 33, 510–517 (2015).

  14. 14.

    et al. Cell 159, 647–661 (2014).

  15. 15.

    et al. Nature 517, 583–588 (2015).

  16. 16.

    , , & Genome Biol. 7, R63 (2006).

  17. 17.

    et al. Proc. Natl. Acad. Sci. USA 106, 8513–8518 (2009).

  18. 18.

    , , & Mol. Endocrinol. 23, 1556–1562 (2009).

  19. 19.

    , , , & Nat. Methods 2, 779–784 (2005).

  20. 20.

    et al. Cell Stem Cell 18, 541–553 (2016).

  21. 21.

    & Cold Spring Harb. Protoc. 2016 (2016).

  22. 22.

    & Cold Spring Harb. Protoc. 2016 (2016).

  23. 23.

    et al. Bioinformatics 31, 3676–3678 (2015).

  24. 24.

    et al. Mol. Cell 32, 735–746 (2008).

  25. 25.

    et al. Cell 136, 952–963 (2009).

  26. 26.

    et al. Nat. Struct. Mol. Biol. 17, 901–908 (2010).

  27. 27.

    et al. Mol. Cell 51, 519–530 (2013).

Download references

Acknowledgements

The authors thank the members of the laboratories of L.S.Q. and N.J.K. for advice and helpful discussions, and the Stanford Functional Genomics Facility and UCSD Sequencing Facility for technical support. We thank D. Zhao from the laboratory of L.S.Q. for advice and help in cloning the sgRNA library. L.S.Q. acknowledges support from the NIH Office of the Director (OD), the National Institute of Dental & Craniofacial Research (NIDCR), the Department of Defense Breast Cancer Research Breakthrough Program, the Pew Charitable Trusts and the Alfred P. Sloan Foundation. D.D. and A.R. acknowledge support from the Quantitative Bioscience Institute (QBI). M.C., N.J.K. and L.S.Q. acknowledge support from the J. David Gladstone Biofulcrum Program. This work was supported by DP5 OD017887, NIH R01 DA036858, NIH U01EB021240, DOD W81XWH-17-1-0018, a Pew Scholar Fellowship and an Alfred P. Sloan Fellowship to L.S.Q., as well as by NIH grants P50 GM082250, U19 AI106754, P01 HL089707, R01 GM084279, U19 AI118610 and R01 AI120694 to N.J.K.

Author information

Author notes

    • Dan Du
    •  & Assen Roguev

    These authors contributed equally to this work.

Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California, USA.

    • Dan Du
    • , Meng Chen
    •  & Lei S Qi
  2. Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

    • Dan Du
    •  & Lei S Qi
  3. Stanford ChEM-H, Stanford University, Stanford, California, USA.

    • Dan Du
    •  & Lei S Qi
  4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.

    • Assen Roguev
    • , David E Gordon
    • , Si-Han Chen
    • , Michael Shales
    •  & Nevan J Krogan
  5. Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, California, USA.

    • Assen Roguev
    • , David E Gordon
    • , Si-Han Chen
    • , Michael Shales
    •  & Nevan J Krogan
  6. J. David Gladstone Institutes, San Francisco, California, USA.

    • Meng Chen
    •  & Nevan J Krogan
  7. Division of Genetics, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

    • John Paul Shen
    •  & Trey Ideker
  8. Department of Bioengineering, University of California, San Diego, La Jolla, California, USA.

    • John Paul Shen
    • , Trey Ideker
    •  & Prashant Mali

Authors

  1. Search for Dan Du in:

  2. Search for Assen Roguev in:

  3. Search for David E Gordon in:

  4. Search for Meng Chen in:

  5. Search for Si-Han Chen in:

  6. Search for Michael Shales in:

  7. Search for John Paul Shen in:

  8. Search for Trey Ideker in:

  9. Search for Prashant Mali in:

  10. Search for Lei S Qi in:

  11. Search for Nevan J Krogan in:

Contributions

L.S.Q. and N.J.K. conceived the project. L.S.Q., N.J.K., D.D. and A.R. designed the experiments; L.S.Q. and D.D. designed the sgRNA library; D.D. constructed the sgRNA libraries and performed library screens; D.D. and A.R. performed sample preparation and sequencing; A.R. analyzed the sequencing data, curated the data sets and scored the data; D.D., S.-H.C. and M.C. performed the CRISPRi validation experiments; D.E.G. performed the esiRNA validation experiments; M.S. contributed to analysis and figure generation; J.P.S., T.I. and P.M. provided technical advice; L.S.Q., N.J.K. and A.R. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Lei S Qi or Nevan J Krogan.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1–8, and Supplementary Notes 1 and 2

Text files

  1. 1.

    Supplementary Data Set 1

    Genetic interactions used to build networks on Figure 2a,b

Excel files

  1. 1.

    Supplementary Data Set 2

    Single sgRNA screens and rapamycin screen raw data

Zip files

  1. 1.

    Supplementary Data Set 3

    Double sgRNA screens raw data and data used in individual figures

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.4286