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A transcription activator-like effector toolbox for genome engineering

Nature Protocols volume 7, pages 171192 (2012) | Download Citation

Abstract

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34–amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

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Acknowledgements

We thank the entire Zhang laboratory for their support. L.C. is supported by a Howard Hughes Medical Institute International Student Research Fellowship. Y.Z. is supported by a Simons Foundation Fellowship. M.M.C. is supported by a Massachusetts Institute of Technology Undergraduate Research Opportunities scholarship. F.Z. is supported by a US National Institutes of Health Transformative R01 and by the McKnight and Simons Foundations, Robert Metcalfe and Michael Boylan.

Author information

Author notes

    • Neville E Sanjana
    • , Le Cong
    •  & Yang Zhou

    These authors contributed equally to this work.

Affiliations

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

    • Neville E Sanjana
    • , Le Cong
    • , Yang Zhou
    • , Margaret M Cunniff
    • , Guoping Feng
    •  & Feng Zhang
  2. Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Neville E Sanjana
    • , Le Cong
    • , Yang Zhou
    • , Margaret M Cunniff
    • , Guoping Feng
    •  & Feng Zhang
  3. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA.

    • Le Cong

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Contributions

N.E.S., L.C., Y.Z. and F.Z. wrote the manuscript. M.M.C. designed the online TALE sequence verification software. F.Z. and G.F. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Feng Zhang.

Supplementary information

Word documents

  1. 1.

    Supplementary Data 1

    Nucleotide sequences of the 4 monomer plasmids, 4 TALE-TF cloning backbone plasmids, and 4 TALEN cloning backbone plasmids.

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DOI

https://doi.org/10.1038/nprot.2011.431

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