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

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.

SUBMITTER: Zhang F 

PROVIDER: S-EPMC3084533 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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

Zhang Feng F   Cong Le L   Lodato Simona S   Kosuri Sriram S   Church George M GM   Arlotta Paola P  

Nature biotechnology 20110119 2

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 typ  ...[more]