title: Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.
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.
authors.affiliation: Division of Chemical and Life Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Lixin
authors.initials: L
authors.lastname: Li
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Piatek
authors.forename: Ahmed
authors.initials: A
authors.lastname: Atef
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Piatek
authors.forename: Anjar
authors.initials: A
authors.lastname: Wibowo
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Fang
authors.forename: J S M
authors.initials: JS
authors.lastname: Sabir
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
authors.forename: Magdy M
authors.initials: MM
authors.lastname: Mahfouz
mesh_terms: D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D014026:Nicotiana / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D004720:Endonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific