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Nat Biotechnol. 2011 Feb;29(2):149-53. doi: 10.1038/nbt.1775. Epub 2011 Jan 19.

Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription.

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  • 1Harvard University, Cambridge, Massachusetts, USA.

Abstract

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.

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PMID:
21248753
[PubMed - indexed for MEDLINE]
PMCID:
PMC3084533
Free PMC Article

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