State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
J Genet Genomics. 2014 Jun 20;41(6):339-47. doi: 10.1016/j.jgg.2014.05.002. Epub 2014 May 17.
Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.
转录激活因子样效应物（TALEs）是与细菌免疫系统相关的一类蛋白，最近被应用于精确的基因靶向这一极有前景的方法中。由于 TALEs 具有重复的特征，现有的 TALE 组装方法要么非常复杂、耗时，要么在普通实验室中很难处理。在这里，我们报道了一种快速、高效、简单的 TALE 组装方法。该方法利用尿嘧啶特异性内切酶（USER），一种可以切割 DNA 构建体并产生长而独特的单链 DNA 突出端的酶。经过 USER 处理，每个 TALE 重复单元上的突出端可以以不依赖连接的方式分层重新连接，形成五聚体。最终，三个五聚体通过 Golden Gate 克隆组装成完整的 TALE 构建体。用这种方法生成的 TALEN 核酸酶（TALENs）在人细胞（如 HEK293FT 细胞）中显示出很高的基因组编辑活性。使用这种方法，我们成功地合成了三对靶向内源性 Tet1 基因座的 TALEN，并证明它们都可以特异性地靶向 Tet1 基因，尽管程度不同。与其他 TALEN 组装方法相比，这种方法的劳动强度要小得多，速度也相当快，仅用两天时间就能以高效率获得阳性克隆。因此，我们为有效 TALENs 的合成提供了一种更简单的方法，这可能极大地促进 TALEN 技术在基因组编辑中的广泛应用，特别是对于需要精确靶向的人类细胞。
