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Methods Mol Biol. 2015;1239:133-59. doi: 10.1007/978-1-4939-1862-1_7.

Efficient design and assembly of custom TALENs using the Golden Gate platform.

Author information

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

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.

PMID:
25408404
DOI:
10.1007/978-1-4939-1862-1_7
[Indexed for MEDLINE]

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