Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochem Biophys Res Commun. 2013 Nov 8;441(1):262-5. doi: 10.1016/j.bbrc.2013.10.060. Epub 2013 Oct 19.
Transcription activator-like effectors (TALEs) are convenient tools for genome engineering at specific genomic sites. However, their use is constrained because most TALE binding sites are preceded by a highly conserved 5' terminal T nucleotide (5'-T). To remove the 5'-T constraint, we substituted tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain for other amino acids. Furthermore, we randomized four amino acid residues of the hairpin loop region of repeat-1. Although point mutation was insufficient to remove the 5'-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5'-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5'-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5'-T constraint and thus allow targeting of any DNA sequences.
转录激活因子样效应物（TALEs）是在特定基因组位点进行基因组工程的便捷工具。然而，由于大多数 TALE 结合位点都以前面有一个高度保守的 5'端 T 核苷酸（5'-T）为特征，因此它们的使用受到限制。为了消除 5'-T 限制，我们用其他氨基酸替代了 dHax3 N 端结构域重复 1 环区的色氨酸 232。此外，我们随机化了重复 1 发夹环区的四个氨基酸残基。尽管点突变不足以消除 5'-T 限制，但来自随机文库的定向进化产生了具有无偏靶向 5'-碱基的重复 1 突变体。我们的结果表明，dHax3 的重复 1 环区对于 5'-碱基容纳很重要，并且 TALEs 的重复 1 的分子进化是一种有效策略，可以消除 5'-T 限制，从而允许靶向任何 DNA 序列。
