Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-12

AUTHORS

Alexandre Juillerat, Marine Beurdeley, Julien Valton, Séverine Thomas, Gwendoline Dubois, Mikhail Zaslavskiy, Jérome Mikolajczak, Fabian Bietz, George H Silva, Aymeric Duclert, Fayza Daboussi, Philippe Duchateau

ABSTRACT

BACKGROUND: The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. RESULTS: In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. CONCLUSION: Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. More... »

PAGES

13

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2199-15-13

DOI

http://dx.doi.org/10.1186/1471-2199-15-13

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1021728343

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24997498


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