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Heritable genome editing in C. elegans via a CRISPR-Cas9 system

Nature Methods volume 10, pages 741–743 (2013)Cite this article

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Abstract

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

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Figure 1: Vectors that drive expression of Cas9 and sgRNAs in C. elegans.
Figure 2: Heritable, targeted gene disruptions in the germ line using CRISPR-Cas systems.

References

  1. Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. Nature 482, 331–338 (2012).

    CAS  Article  Google Scholar 

  2. Terns, M.P. & Terns, R.M. Curr. Opin. Microbiol. 14, 321–327 (2011).

    CAS  Article  Google Scholar 

  3. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  5. Dicarlo, J.E. et al. Nucleic Acids Res. 41, 4336–4343 (2013).

    CAS  Article  Google Scholar 

  6. Gratz, S.J. et al. Genetics advance online publication 24 May 2013 (doi: 10.1534/genetics.113.152710).

    CAS  Article  Google Scholar 

  7. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Nat. Biotechnol. 31, 230–232 (2013).

    CAS  Article  Google Scholar 

  8. Cong, L. et al. Science 339, 819–823 (2013).

    CAS  Article  Google Scholar 

  9. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    Article  Google Scholar 

  10. Mali, P. et al. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  11. Hwang, W.Y. et al. Nat. Biotechnol. 31, 227–229 (2013).

    CAS  Article  Google Scholar 

  12. Wang, H. et al. Cell 153, 410–418 (2013).

    Google Scholar 

  13. Frokjaer-Jensen, C., Davis, M.W., Ailion, M. & Jorgensen, E.M. Nat. Methods 9, 117–118 (2012).

    CAS  Article  Google Scholar 

  14. Miyagishi, M. & Taira, K. Nat. Biotechnol. 20, 497–500 (2002).

    CAS  Article  Google Scholar 

  15. Fruscoloni, P., Zamboni, M., Panetta, G., De Paolis, A. & Tocchini-Valentini, G.P. Nucleic Acids Res. 23, 2914–2918 (1995).

    CAS  Article  Google Scholar 

  16. Zecherle, G.N., Whelen, S. & Hall, B.D. Mol. Cell Biol. 16, 5801–5810 (1996).

    CAS  Article  Google Scholar 

  17. von Mende, N., Bird, D.M., Albert, P.S. & Riddle, D.L. Cell 55, 567–576 (1988).

    CAS  Article  Google Scholar 

  18. Maduro, M. & Pilgrim, D. Genetics 141, 977–988 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. EMBO J. 10, 3959–3970 (1991).

    CAS  Article  Google Scholar 

  20. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  Article  Google Scholar 

  21. Brenner, S. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Thomas, J., Lea, K., Zucker-Aprison, E. & Blumenthal, T. Nucleic Acids Res. 18, 2633–2642 (1990).

    CAS  Article  Google Scholar 

  23. Meyer, L.R. et al. Nucleic Acids Res. 41, D64–D69 (2013).

    CAS  Article  Google Scholar 

  24. Gibson, D.G. et al. Nat. Methods 6, 343–345 (2009).

    CAS  Article  Google Scholar 

  25. Kadandale, P., Chatterjee, I. & Singson, A. Methods Mol. Biol. 518, 123–133 (2009).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank members of the Caenorhabditis Genetics Center for providing the N2 strain used in our experiments, and B. Stern, A. Murray, A. Saltzman, Joe Calarco and members of the Calarco laboratory for comments on the manuscript. This work was supported by US National Institutes of Health Early Independence Award (1DP5OD009153) and additional support from Harvard University to J.A.C., by National Institutes of Health grant R01GM072551 to M.P.C., and a National Human Genome Research Institute Center of Excellence in Genome Sciences award to G.M.C. A.E.F. is supported by a Ralph Ellison/American Federation for Aging Research postdoctoral fellowship.

Author information

Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Ari E Friedland, Yonatan B Tzur, Monica P Colaiácovo & George M Church

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA

    Kevin M Esvelt & George M Church

  3. Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA

    John A Calarco

Authors
  1. Ari E Friedland
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  2. Yonatan B Tzur
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  3. Kevin M Esvelt
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  4. Monica P Colaiácovo
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  5. George M Church
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  6. John A Calarco
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Contributions

A.E.F., K.M.E. and J.A.C. conceived of and designed experiments, with help from Y.B.T.; A.E.F. and J.A.C. assembled vectors; A.E.F. and J.A.C. performed microinjections and screened mutants; A.E.F., J.A.C. and Y.B.T. performed off-target genotyping analysis; A.E.F., K.M.E. and J.A.C. wrote the manuscript with input from Y.B.T., M.P.C. and G.M.C.

Corresponding authors

Correspondence to George M Church or John A Calarco.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–4, and Supplementary Tables 1 and 2 (PDF 5711 kb)

Supplementary Video 1

Movie of a wild-type (N2 strain) worm. (MOV 1114 kb)

Supplementary Video 2

Movie of an unc-119 worm created by Cas9-mediated gene disruption. (MOV 1739 kb)

Supplementary Video 3

Movie of a dpy-13 worm created by Cas9-mediated gene disruption. (MOV 1641 kb)

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Friedland, A., Tzur, Y., Esvelt, K. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10, 741–743 (2013). https://doi.org/10.1038/nmeth.2532

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