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.
Harvard University, Cambridge, Massachusetts, USA.
Feng
F
Zhang
Le
L
Cong
Simona
S
Lodato
Sriram
S
Kosuri
George M
GM
Church
Paola
P
Arlotta
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D055748:SOXB1 Transcription Factors / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014974:Xanthomonas / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; D004247:DNA
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