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Assembly of custom TALE-type DNA binding domains by modular cloning
Open accessAbstract
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.
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Citations
Cited by
TAL effectors: customizable proteins for DNA targeting
Bogdanove, A. J., & Voytas, D. F. (2011) · Science · doi:10.1126/science.1204094 · Cited by 984
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity
Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T., & Cathomen, T. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr597 · Cited by 714
Self-citationGenome Engineering with Targetable Nucleases
Carroll, D. (2014) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-060713-035418 · Cited by 568
GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules.
Sarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011) · PLoS ONE · doi:10.1371/journal.pone.0021622 · Cited by 348
Bloom, K., Ely, A., Mussolino, C., Cathomen, T., & Arbuthnot, P. (2013) · Molecular Therapy · doi:10.1038/mt.2013.170 · Cited by 217
Metadata
- Authors
- Morbitzer, R., Elsaesser, J., Hausner, J., & Lahaye, T.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2011-03-18
- Volume
- 39
- Issue
- 13
- Pages
- 5790-5799
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2002302294
- Cited by
- 185
- References
- 0
- FWCI
- 11.29
- Type
- journal article
- Language
- en
- Open access
- gold
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- Morbitzer, R., Elsaesser, J., Hausner, J., & Lahaye, T. (2011). Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Research. https://doi.org/10.1093/nar/gkr151
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