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...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21625552/?tool=EBI |
| _version_ | 1819036828312797184 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-21T08:11:43Z |
| format | Article |
| id | doaj.art-c54548b79ce44e05b14f695fea016465 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T08:11:43Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c54548b79ce44e05b14f695fea0164652022-12-21T19:10:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0165e1972210.1371/journal.pone.0019722Assembly of designer TAL effectors by Golden Gate cloning.Ernst WeberRamona GruetznerStefan WernerCarola EnglerSylvestre MarillonnetGeneration 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21625552/?tool=EBI |
| spellingShingle | Ernst Weber Ramona Gruetzner Stefan Werner Carola Engler Sylvestre Marillonnet Assembly of designer TAL effectors by Golden Gate cloning. PLoS ONE |
| title | 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_short | Assembly of designer TAL effectors by Golden Gate cloning. |
| title_sort | assembly of designer tal effectors by golden gate cloning |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21625552/?tool=EBI |
| work_keys_str_mv | AT ernstweber assemblyofdesignertaleffectorsbygoldengatecloning AT ramonagruetzner assemblyofdesignertaleffectorsbygoldengatecloning AT stefanwerner assemblyofdesignertaleffectorsbygoldengatecloning AT carolaengler assemblyofdesignertaleffectorsbygoldengatecloning AT sylvestremarillonnet assemblyofdesignertaleffectorsbygoldengatecloning |