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A transcription activator-like effector toolbox for genome engineering

Nature Protocols volume 7, pages 171–192 (2012)Cite this article

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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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Figure 1: A TALE toolbox for genome engineering.
Figure 2: Timeline for the construction of TALE-TFs and TALENs.
Figure 3: Construction of TALE DNA-binding domains using hierarchical ligation assembly.
Figure 4: PCR plate setup used to generate a plate of monomers for constructing custom 18-mer TALE DNA-binding domains.
Figure 5: Example gel results from the TALE construction procedure.
Figure 6: Examples of TALE-TF and TALEN activity in 293FT cells.

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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.

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Author notes

  1. Neville E Sanjana, Le Cong and Yang Zhou: These authors contributed equally to this work.

Authors and 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.

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Correspondence to Feng Zhang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

Nucleotide sequences of the 4 monomer plasmids, 4 TALE-TF cloning backbone plasmids, and 4 TALEN cloning backbone plasmids. (DOC 67 kb)

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Sanjana, N., Cong, L., Zhou, Y. et al. A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7, 171–192 (2012). https://doi.org/10.1038/nprot.2011.431

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