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    De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

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    Type
    Article
    Authors
    Mahfouz, Magdy M. cc
    Li, Lixin
    Shamimuzzaman, Md.
    Wibowo, Anjar Tri
    Fang, Xiaoyun
    Zhu, Jian-Kang 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
    2011-01-24
    Online Publication Date
    2011-01-24
    Print Publication Date
    2011-02-08
    Permanent link to this record
    http://hdl.handle.net/10754/561706
    
    Metadata
    Show full item record
    Abstract
    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.
    Citation
    Mahfouz, M. M., Li, L., Shamimuzzaman, M., Wibowo, A., Fang, X., & Zhu, J.-K. (2011). De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proceedings of the National Academy of Sciences, 108(6), 2623–2628. doi:10.1073/pnas.1019533108
    Publisher
    Proceedings of the National Academy of Sciences
    Journal
    Proceedings of the National Academy of Sciences
    DOI
    10.1073/pnas.1019533108
    PubMed ID
    21262818
    PubMed Central ID
    PMC3038751
    Additional Links
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038751
    ae974a485f413a2113503eed53cd6c53
    10.1073/pnas.1019533108
    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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