Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, USA.
Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431.
Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.
转录激活因子样效应物 (TALEs) 是一类天然存在于植物病原体黄单胞菌中的 DNA 结合蛋白。每个 TALE 的 DNA 结合结构域由串联的 34 个氨基酸重复模块组成，这些模块可以根据一个简单的密码进行重排，以靶向新的 DNA 序列。定制的 TALEs 可用于多种基因组工程应用，包括转录调控和基因组编辑。在这里，我们描述了一个使用层次连接程序快速构建定制 TALE 转录因子 (TALE-TF) 和核酸酶 (TALEN) 的工具包。该工具包可在 1 周内实现定制 TALE-TF 和 TALEN 的经济高效和快速构建，并可轻松扩展到并行构建多个靶标 TALEs。我们还提供了使用定量逆转录 PCR 和 Surveyor 核酸酶分别在哺乳动物细胞中测试定制 TALE-TF 和 TALEN 活性的详细信息。本文描述的 TALE 工具包将能够实现广泛的生物学应用。
