College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaan'xi, PR China.
PLoS One. 2013 Jun 20;8(6):e66459. doi: 10.1371/journal.pone.0066459. Print 2013.
DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.
来自黄单胞菌的转录激活样效应因子（TALEs）的 DNA 结合域由串联重复组成，可以根据简单的密码进行重排，从而靶向具有高度 DNA 结合特异性的新 DNA 序列。这项技术已成功应用于多种物种的基因组工程。然而，组装长 TALE 串联重复仍然是一个巨大的挑战，限制了这项技术的广泛应用。尽管最近有几种新的方法可用于有效地组装 TALE 重复，但它们都需要复杂的设备或熟练的技术人员来进行操作。在这里，我们描述了一种基于 TALE 重复四聚体文库生成定制 TALE 核酸酶（TALEN）和 TALE 转录因子（TALE-TF）的简单而有效的方法。由 256 个四聚体组成的四聚体文库涵盖了所有可能的 4 碱基对组合。设计了一组独特的引物用于扩增这些四聚体。通过一步消化/连接反应进行 PCR 产物的组装。使用我们的方法生成了 12 个 TALE 构建体，包括 4 对靶向小鼠 Gt(ROSA)26Sor 基因和小鼠 Mstn 基因序列的 TALEN，以及 4 个靶向小鼠 Oct4、c-Myc、Klf4 和 Sox2 基因启动子序列的 TALE-TF 构建体。构建例程耗时 3 天，可并行构建。在菌落 PCR 验证中，阳性克隆的比例平均为 64%。测序结果表明，所有 TALE 构建体都具有很高的成功率。这是一种使用最常见的酶和设备的快速且具有成本效益的方法，成功率很高。
