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Continuous directed evolution of DNA-binding proteins to improve TALEN specificity

Nature Methods volume 12, pages 939–942 (2015)Cite this article

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Abstract

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator–like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

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Figure 1: Development of DB-PACE and its application to TALEs.
Figure 2: High-throughput specificity profiling of canonical and evolved TALENs.

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Acknowledgements

This work was supported by Defense Advanced Research Projects Agency (DARPA) HR0011-11-2-0003, DARPA N66001-12-C-4207, a grant from the US National Institutes of Health (NIH)/NIGMS (R01 GM095501 and R01 GM065400) and the Howard Hughes Medical Institute. S.Q.T., J.D.S. and J.K.J. were supported by an NIH Director's Pioneer Award (DP1 GM105378). S.Q.T. was supported by NIH F32 GM105189. A.H.B. was supported by the Harvard Chemical Biology Program and a US National Science Foundation Graduate Research Fellowship.

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Author notes

  1. Jeffry D Sander

    Present address: Present address: DuPont Pioneer, Johnston, Iowa, USA.,

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Basil P Hubbard, Ahmed H Badran, John A Zuris, John P Guilinger, Kevin M Davis, Liwei Chen & David R Liu

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

    Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

  3. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

  4. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

  6. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    David R Liu

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  1. Basil P Hubbard
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Contributions

B.P.H. designed the research, performed experiments, analyzed data and wrote the manuscript. A.H.B. assisted in the design of the one-hybrid system, and A.H.B. and K.M.D. contributed materials and performed experiments. J.A.Z., J.P.G. and L.C. performed experiments and data analysis. S.Q.T. prepared materials for TALEN cleavage analysis in cells. J.D.S. contributed experimentally validated TALE arrays. D.R.L. designed and supervised the research and wrote the manuscript. All of the authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R Liu.

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Competing interests

D.R.L. is a consultant for Editas Medicine. J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics 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.

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Supplementary Figures 1–25, Supplementary Tables 1–4, Supplementary Results, Supplementary Discussion and Supplementary Notes 1–4 (PDF 14767 kb)

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Hubbard, B., Badran, A., Zuris, J. et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nat Methods 12, 939–942 (2015). https://doi.org/10.1038/nmeth.3515

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