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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Binding Sites
  • Cell Line
  • DNA / genetics
  • DNA / metabolism
  • Genetic Engineering*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / genetics
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • SOXB1 Transcription Factors / genetics
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic*
  • Xanthomonas / genetics
  • Xanthomonas / metabolism

Substances

  • Bacterial Proteins
  • KLF4 protein, human
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • Transcription Factors
  • DNA