Brief Communication | Published:

Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects

Nature Methods volume 11, pages 399402 (2014) | Download Citation

Abstract

Bacterial RNA–directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.

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.

    , , , & Nat. Rev. Genet. 11, 636–646 (2010).

  2. 2.

    & Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).

  3. 3.

    , & Nature 482, 331–338 (2012).

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    et al. Nat. Biotechnol. 31, 227–229 (2013).

  8. 8.

    , , & Nat. Biotechnol. 31, 230–232 (2013).

  9. 9.

    , , , & Nat. Biotechnol. 31, 233–239 (2013).

  10. 10.

    et al. Elife 2, e00471 (2013).

  11. 11.

    et al. Cell Res. 23, 720–723 (2013).

  12. 12.

    et al. Cell 152, 1173–1183 (2013).

  13. 13.

    et al. Cell 153, 910–918 (2013).

  14. 14.

    et al. Cell 154, 1370–1379 (2013).

  15. 15.

    et al. Nat. Biotechnol. 31, 822–826 (2013).

  16. 16.

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

  17. 17.

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

  18. 18.

    et al. Nat. Biotechnol. 31, 839–843 (2013).

  19. 19.

    et al. Genome Res. 22, 1316–1326 (2012).

  20. 20.

    et al. Genome Res. 22, 1327–1333 (2012).

  21. 21.

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

  22. 22.

    et al. Proc. Natl. Acad. Sci. USA 99, 13498–13503 (2002).

  23. 23.

    et al. Nat. Biotechnol. 29, 695–696 (2011).

  24. 24.

    Nat. Rev. Genet. 9, 619–631 (2008).

  25. 25.

    , , & J. Cell Biol. 146, 905–916 (1999).

  26. 26.

    & Bioinformatics 26, 841–842 (2010).

Download references

Acknowledgements

We thank members of the entire Huang laboratory for their support and advice, G. Tischler for the C++ alignment code, and J. Liu and C. Gao for help with deep sequencing. This work was supported by grants from the 973 program (2010CB945101 and 2011CB944301) and a core grant from the Wellcome Trust.

Author information

Author notes

    • Bin Shen
    • , Wensheng Zhang
    •  & Jun Zhang

    These authors contributed equally to this work.

Affiliations

  1. Ministry of Education Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, China.

    • Bin Shen
    • , Jun Zhang
    • , Jiankui Zhou
    • , Jianying Wang
    • , Li Chen
    •  & Xingxu Huang
  2. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

    • Wensheng Zhang
    • , Alex Hodgkins
    • , Vivek Iyer
    •  & William C Skarnes
  3. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.

    • Lu Wang

Authors

  1. Search for Bin Shen in:

  2. Search for Wensheng Zhang in:

  3. Search for Jun Zhang in:

  4. Search for Jiankui Zhou in:

  5. Search for Jianying Wang in:

  6. Search for Li Chen in:

  7. Search for Lu Wang in:

  8. Search for Alex Hodgkins in:

  9. Search for Vivek Iyer in:

  10. Search for Xingxu Huang in:

  11. Search for William C Skarnes in:

Contributions

B.S., W.Z., J. Zhang, X.H. and W.C.S. designed the experiments and wrote the manuscript. B.S., W.Z., J. Zhang, J. Zhou, J.W. and L.C. performed the experiments. L.W., A.H. and V.I. performed the computational analysis of off-target sites.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xingxu Huang or William C Skarnes.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Tables 1–14 and Supplementary Note

Excel files

  1. 1.

    Supplementary Data

    Paired off-target sites for AR-A and AR-B sgRNAs. Ten closest paired sites in the mouse genome with similar sequence to each combination of AR-A and AR-B Cas9 target sites.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.2857

Further reading