Brief Communication | Published:

Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease

Nature Biotechnology volume 33, pages 11591161 (2015) | Download Citation

  • A Corrigendum to this article was published on 07 April 2016

This article has been updated

Abstract

We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 04 February 2016

    In the version of this article initially published, when discussing the data in Figure 2b, on p. 1160, we wrote, “...targeting the same HBG1/2 promoter and found they had 32 and 55 perturbed transcripts....” This should have been “31 and 55 perturbed transcripts” as in the sentence in the figure legend discussing the same data. The error has been corrected in the HTML and PDF versions of the article.

Accessions

Primary accessions

BioProject

Referenced accessions

BioProject

References

  1. 1.

    et al. Cell 160, 339–350 (2015).

  2. 2.

    et al. Nat. Methods 10, 1116–1121 (2013).

  3. 3.

    et al. Science 337, 816–821 (2012).

  4. 4.

    , , & Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

  5. 5.

    et al. Nat. Methods 10, 973–976 (2013).

  6. 6.

    et al. Nat. Biotechnol. 31, 833–838 (2013).

  7. 7.

    et al. Nat. Methods 10, 977–979 (2013).

  8. 8.

    et al. Nature 500, 472–476 (2013).

  9. 9.

    et al. Cell 154, 442–451 (2013).

  10. 10.

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

  11. 11.

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

  12. 12.

    et al. Cell 156, 935–949 (2014).

  13. 13.

    et al. Nat. Biotechnol. 31, 827–832 (2013).

  14. 14.

    et al. Mol. Cell 56, 333–339 (2014).

  15. 15.

    et al. Nat. Biotechnol. 32, 670–676 (2014).

  16. 16.

    et al. Science 343, 84–87 (2014).

  17. 17.

    et al. Cell 159, 440–455 (2014).

  18. 18.

    et al. Nat. Biotechnol. 33, 390–394 (2015).

  19. 19.

    , , , & Nat. Biotechnol. 32, 279–284 (2014).

  20. 20.

    et al. Nat. Methods. (7 September 2015).

  21. 21.

    et al. Cell 154, 1380–1389 (2013).

  22. 22.

    & BMC Bioinformatics 12, 323 (2011).

Download references

Acknowledgements

The authors thank K. Zheng and I. Slaymaker for their support and input. J.E.D. is supported by a Life Science Research Foundation post-doctoral fellowship of the Cystic Fibrosis Foundation. O.O.A. is supported by a Friends of the McGovern Institute Fellowship. J.S.G. is supported by a Department of Energy (DOE) Computational Science Graduate Fellowship. F.Z. is supported by the National Institute of Mental Health (NIMH) (1DP1-MH100706), the Poitras, Vallee, Simons, Paul G. Allen and New York Stem Cell Foundations, and David R. Cheng and Bob Metcalfe. The authors plan to make the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang laboratory website (http://www.genome-engineering.org/).

Author information

Author notes

    • James E Dahlman
    •  & Omar O Abudayyeh

    These authors contributed equally to this work.

Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • James E Dahlman
    • , Omar O Abudayyeh
    • , Julia Joung
    • , Jonathan S Gootenberg
    • , Feng Zhang
    •  & Silvana Konermann
  2. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.

    • James E Dahlman
  3. McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA.

    • Omar O Abudayyeh
    • , Julia Joung
    • , Jonathan S Gootenberg
    • , Feng Zhang
    •  & Silvana Konermann
  4. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Omar O Abudayyeh
    • , Julia Joung
    • , Jonathan S Gootenberg
    • , Feng Zhang
    •  & Silvana Konermann
  5. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Omar O Abudayyeh
    • , Julia Joung
    • , Jonathan S Gootenberg
    • , Feng Zhang
    •  & Silvana Konermann
  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Jonathan S Gootenberg

Authors

  1. Search for James E Dahlman in:

  2. Search for Omar O Abudayyeh in:

  3. Search for Julia Joung in:

  4. Search for Jonathan S Gootenberg in:

  5. Search for Feng Zhang in:

  6. Search for Silvana Konermann in:

Contributions

J.E.D., O.O.A., F.Z. and S.K. conceived this study and designed the experiments. J.E.D., O.O.A., S.K., J.J. and J.S.G. performed experiments. J.E.D., O.O.A., F.Z. and S.K. wrote the manuscript with input from all authors.

Competing interests

J.E.D., O.O.A., F.Z. and S.K. have filed for intellectual property rights related to material described in this publication.

Corresponding authors

Correspondence to James E Dahlman or Silvana Konermann.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text

    Supplementary Figures 1-4 and Supplementary Notes

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.3390

Further reading