A transcription activator-like effector toolbox for genome engineering

Nature Protocols

Volume 7 Issue 1, , Pages 171 - 192

doi: 10.1038/nprot.2011.431

Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.


, , , , ,

a Broad Institute of Massachusetts Institute of Technology and Harvard

b Department of Brain and Cognitive Sciences, McGovern Institute for Brain Research, Massachusetts Institute of Technology

c Program in Biological and Biomedical Sciences, Harvard Medical School


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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