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

Abstract

TALEs (transcription activator–like effectors) contain a large number of nearly identical repeats, which makes it difficult to synthesize new variants. Feng et al. describe a facile method for assembling TALEs and show TALEs' utility for activating expression of endogenous human genes. 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.