title: Assembly of custom TALE-type DNA binding domains by modular cloning.
abstract: Transcription activator-like effector (TALE) DNA binding proteins show tremendous potential as molecular tools for targeted binding to any desired DNA sequence. Their DNA binding domain consists of tandem arranged repeats, and due to this repetitive structure it is challenging to generate designer TALEs (dTALEs) with user-defined specificity. We present a cloning approach that facilitates the assembly of multiple repeat-encoding DNA fragments that translate into dTALEs with pre-defined DNA binding specificity. This method makes use of type IIS restriction enzymes in two sequential cut-ligase reactions to build dTALE repeat arrays. We employed this modular approach for generation of a dTALE that differentiates between two highly similar DNA sequences that are both targeted by the Xanthomonas TALE, AvrBs3. These data show that this modular assembly system allows rapid generation of highly specific TALE-type DNA binding domains that target binding sites of predefined length and sequence. This approach enables the rapid and flexible production of dTALEs for gene regulation and genome editing in routine and high-throughput applications.
authors.affiliation: Institute of Biology, Genetics, University of Munich (LMU), 82152 Martinsried and Institute of Biology, Martin-Luther-University, 06099 Halle, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Morbitzer
authors.forename: Janett
authors.initials: J
authors.lastname: Elsaesser
authors.forename: Jens
authors.initials: J
authors.lastname: Hausner
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D005822:Genetic Vectors; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D015534:Trans-Activators; D004247:DNA; C483995:BsaI endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific