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

Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription 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. Systematic interrogation and engineering of biological systems in normal and pathological states depend on the ability to manipulate the genome of target cells with efficiency and precision . Some naturally occurring DNA-binding proteins have been engineered to enable sequence-specific DNA perturbation, including polydactyl zinc fingers and meganucleases . In particular, engineered zinc fingers can be attached to a wide variety of effector domains such as nucleases, transcription effectors and http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2011 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
ISSN
1087-0156
eISSN
1546-1696
D.O.I.
10.1038/nbt.1775
Publisher site
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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. Systematic interrogation and engineering of biological systems in normal and pathological states depend on the ability to manipulate the genome of target cells with efficiency and precision . Some naturally occurring DNA-binding proteins have been engineered to enable sequence-specific DNA perturbation, including polydactyl zinc fingers and meganucleases . In particular, engineered zinc fingers can be attached to a wide variety of effector domains such as nucleases, transcription effectors and

Journal

Nature BiotechnologySpringer Journals

Published: Jan 19, 2011

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