Article | Published:

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

Nature Biotechnology volume 32, pages 569576 (2014) | Download Citation

Abstract

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

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Acknowledgements

We thank Y. Fu and M. Maeder for helpful discussions and Y. Fu, J. Angstman and B. Kleinstiver for comments on the manuscript. This work was funded by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378), NIH R01 GM088040, NIH P50 HG005550, NIH R01 AR063070, and the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. S.Q.T. was supported by NIH F32 GM105189. This material is based upon work supported by, or in part by, the US Army Research Laboratory and the US Army Research Office under grant number W911NF-11-2-0056. Plasmids described in this work will be deposited with and made available through the nonprofit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas).

Author information

Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nicolas Wyvekens
    • , Cyd Khayter
    • , Jennifer A Foden
    • , Vishal Thapar
    • , Deepak Reyon
    • , Mathew J Goodwin
    • , Martin J Aryee
    •  & J Keith Joung
  2. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nicolas Wyvekens
    • , Cyd Khayter
    • , Jennifer A Foden
    • , Vishal Thapar
    • , Deepak Reyon
    • , Mathew J Goodwin
    • , Martin J Aryee
    •  & J Keith Joung
  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nicolas Wyvekens
    • , Cyd Khayter
    • , Jennifer A Foden
    • , Deepak Reyon
    • , Mathew J Goodwin
    •  & J Keith Joung
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Deepak Reyon
    • , Martin J Aryee
    •  & J Keith Joung

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Contributions

S.Q.T., N.W. and J.K.J. conceived of and designed experiments. S.Q.T., N.W., C.K., J.A.F. and M.J.G. performed experiments. D.R. developed the updated version of the ZiFiT Targeter software, and V.T. and M.J.A. wrote the software program for identifying potential RFN off-target sites. S.Q.T., N.W. and J.K.J. wrote the paper.

Competing interests

J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. and S.Q.T. are inventors on patent applications describing the FokI-dCas9 technology and the multiplex gRNA expression method.

Corresponding author

Correspondence to J Keith Joung.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Discussion and Supplementary Tables 2–6

Excel files

  1. 1.

    Supplementary Table 1

    85 gRNA pairs targeted to the EGFP reporter gene

  2. 2.

    Supplementary Table 7

    Primers and RFN target sites used in this study

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DOI

https://doi.org/10.1038/nbt.2908

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