Plant Molecular Biology

, Volume 78, Issue 4, pp 407–416

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

Authors

  • Lixin Li
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
  • Marek J. Piatek
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
  • Ahmed Atef
    • Department of Biological Sciences, Faculty of SciencesKing Abdulaziz University
  • Agnieszka Piatek
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
  • Anjar Wibowo
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
  • Xiaoyun Fang
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
  • J. S. M. Sabir
    • Department of Biological Sciences, Faculty of SciencesKing Abdulaziz University
    • Department of Horticulture and Landscape ArchitecturePurdue University
    • Division of Chemical and Life Sciences and EngineeringKing Abdullah University of Science and Technology
Article

DOI: 10.1007/s11103-012-9875-4

Cite this article as:
Li, L., Piatek, M.J., Atef, A. et al. Plant Mol Biol (2012) 78: 407. doi:10.1007/s11103-012-9875-4

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.

Keywords

Genome engineeringTALE-based activators and repressorsTALE nucleases (TALENs)Targeted mutagenesisTargeted activation and repressionGenome modifications

Supplementary material

11103_2012_9875_MOESM1_ESM.doc (176 kb)
Supplementary material 1 (DOC 176 kb)
11103_2012_9875_MOESM2_ESM.pdf (70 kb)
Supplementary material 2 (PDF 69 kb)
11103_2012_9875_MOESM3_ESM.pdf (960 kb)
Supplementary material 3 (PDF 960 kb)

Copyright information

© Springer Science+Business Media B.V. 2012