title: Assembly of designer TAL effectors by Golden Gate cloning.
abstract: Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
authors.affiliation: Icon Genetics GmbH, Halle/Saale, Germany.
authors.forename: Ernst
authors.initials: E
authors.lastname: Weber
authors.forename: Ramona
authors.initials: R
authors.lastname: Gruetzner
authors.forename: Stefan
authors.initials: S
authors.lastname: Werner
authors.forename: Carola
authors.initials: C
authors.lastname: Engler
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Marillonnet
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D018745:Genome, Plant; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA