Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China.
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Genes (Basel). 2021 Nov 5;12(11):1761. doi: 10.3390/genes12111761.
Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.
转录激活因子样效应物（TALEs）已被有效地用于靶向基因组编辑、转录调控、表观遗传修饰和特定基因座的 DNA 成像。然而，随着成簇规律间隔短回文重复序列/Cas9 系统的出现，这种具有与 TALEs 相同功能的易于使用的工具已经取代了 TALEs，因为 TALEs 复杂、耗时且在普通实验室中难以操作。在这里，我们描述了一种基于简并密码子的 TALE 组装系统，用于简单、快速和高效的 TALE 组装。通过 PCR 扩增具有非重复 DNA 序列的 TALE 三聚体，并通过 Gibson 组装依次进行组装。我们的方法具有成本效益，仅需要常用的基本分子生物学试剂，并且从目标序列分析到完成仅需 2 小时。这种简单、快速且对实验室友好的 TALE 组装方法将恢复 TALEs 在 DNA 靶向中的价值。
