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Efficient Design and Assembly of Custom TALENs Using the Golden Gate Platform

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Chromosomal Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1239))

Abstract

An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other sequence-specific nucleases. In this chapter, we describe a rapid, inexpensive, and user-friendly protocol for custom TALEN construction based on one of the most popular TALEN assembly platforms, the Golden Gate cloning method. Using this protocol, ready-to-use TALENs with specificity for targets 13–32 bp long are constructed within 5 days.

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Acknowledgment

We thank Rebecca Greenstein for critical reading of the manuscript and helpful comments.

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Authors and Affiliations

  1. Department of Genetics, Cell Biology & Development, University of Minnesota, 350 Cargill Building, 1500 Gortner Avenue, St. Paul, MN, 55108-1023, USA

    Tomas Cermak, Colby G. Starker & Daniel F. Voytas

  2. Center for Genome Engineering, University of Minnesota, 328 Cargill Building, 1500 Gortner Avenue, St. Paul, MN, 55108-1023, USA

    Tomas Cermak, Colby G. Starker & Daniel F. Voytas

Authors
  1. Tomas Cermak
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  2. Colby G. Starker
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  3. Daniel F. Voytas
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Corresponding author

Correspondence to Daniel F. Voytas .

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Editors and Affiliations

  1. Department of Genetics, Genome Engineering and iPSC Center, Washington University School of Medicine, St. Louis, Missouri, USA

    Shondra M. Pruett-Miller

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Cermak, T., Starker, C.G., Voytas, D.F. (2015). Efficient Design and Assembly of Custom TALENs Using the Golden Gate Platform. In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_7

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  • DOI: https://doi.org/10.1007/978-1-4939-1862-1_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1861-4

  • Online ISBN: 978-1-4939-1862-1

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