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    Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

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    Type
    Article
    Authors
    Li, Lixin
    Piatek, Marek J. cc
    Atef, Ahmed
    Piatek, Agnieszka Anna cc
    Wibowo, Anjar Tri
    Fang, Xiaoyun
    Sabir, Jamal Sabir M
    Zhu, Jian-Kang cc
    Mahfouz, Magdy M. cc
    KAUST Department
    Biological and Environmental Science and Engineering (BESE) Division
    Bioscience Program
    Center for Desert Agriculture
    Laboratory for Genome Engineering
    Plant Science
    Plant Science Program
    Date
    2012-01-21
    Online Publication Date
    2012-01-22
    Print Publication Date
    2012-03
    Embargo End Date
    2013-01-21
    Permanent link to this record
    http://hdl.handle.net/10754/562063
    
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    Abstract
    Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.
    Citation
    Li, L., Piatek, M. J., Atef, A., Piatek, A., Wibowo, A., Fang, X., … Mahfouz, M. M. (2012). Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification. Plant Molecular Biology, 78(4-5), 407–416. doi:10.1007/s11103-012-9875-4
    Publisher
    Springer Nature
    Journal
    Plant Molecular Biology
    DOI
    10.1007/s11103-012-9875-4
    PubMed ID
    22271303
    PubMed Central ID
    PMC3580834
    Additional Links
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580834
    http://europepmc.org/articles/pmc3580834?pdf=render
    ae974a485f413a2113503eed53cd6c53
    10.1007/s11103-012-9875-4
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program; Plant Science Program; Center for Desert Agriculture

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