Assembly of designer TAL effectors by Golden Gate cloning.

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 t...

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Autores principales:	Ernst Weber, Ramona Gruetzner, Stefan Werner, Carola Engler, Sylvestre Marillonnet
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c54548b79ce44e05b14f695fea016465
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spelling	oai:doaj.org-article:c54548b79ce44e05b14f695fea0164652021-11-18T06:53:39ZAssembly of designer TAL effectors by Golden Gate cloning.1932-620310.1371/journal.pone.0019722https://doaj.org/article/c54548b79ce44e05b14f695fea0164652011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21625552/?tool=EBIhttps://doaj.org/toc/1932-6203Generation 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.Ernst WeberRamona GruetznerStefan WernerCarola EnglerSylvestre MarillonnetPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 5, p e19722 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ernst Weber Ramona Gruetzner Stefan Werner Carola Engler Sylvestre Marillonnet Assembly of designer TAL effectors by Golden Gate cloning.
description	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.
format	article
author	Ernst Weber Ramona Gruetzner Stefan Werner Carola Engler Sylvestre Marillonnet
author_facet	Ernst Weber Ramona Gruetzner Stefan Werner Carola Engler Sylvestre Marillonnet
author_sort	Ernst Weber
title	Assembly of designer TAL effectors by Golden Gate cloning.
title_short	Assembly of designer TAL effectors by Golden Gate cloning.
title_full	Assembly of designer TAL effectors by Golden Gate cloning.
title_fullStr	Assembly of designer TAL effectors by Golden Gate cloning.
title_full_unstemmed	Assembly of designer TAL effectors by Golden Gate cloning.
title_sort	assembly of designer tal effectors by golden gate cloning.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/c54548b79ce44e05b14f695fea016465
work_keys_str_mv	AT ernstweber assemblyofdesignertaleffectorsbygoldengatecloning AT ramonagruetzner assemblyofdesignertaleffectorsbygoldengatecloning AT stefanwerner assemblyofdesignertaleffectorsbygoldengatecloning AT carolaengler assemblyofdesignertaleffectorsbygoldengatecloning AT sylvestremarillonnet assemblyofdesignertaleffectorsbygoldengatecloning
_version_	1718424254945427456

Assembly of designer TAL effectors by Golden Gate cloning.

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