De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks
Type
ArticleKAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Program
Desert Agriculture Initiative
Laboratory for Genome Engineering
Plant Science
Plant Science Program
Date
2011-01-24Online Publication Date
2011-01-24Print Publication Date
2011-02-08Permanent link to this record
http://hdl.handle.net/10754/561706
Metadata
Show full item recordAbstract
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.1019533108PubMed ID
21262818PubMed Central ID
PMC3038751Additional Links
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038751ae974a485f413a2113503eed53cd6c53
10.1073/pnas.1019533108
Scopus Count
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