FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.
FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.
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DOI：
10.1089/hum.2015.172
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发表时间：
2016-06
影响因子：
4.2
通讯作者：
Ekker SC
中科院分区：
文献类型：
--
作者：
Ma AC;McNulty MS;Poshusta TL;Campbell JM;Martínez-Gálvez G;Argue DP;Lee HB;Urban MD;Bullard CE;Blackburn PR;Man TK;Clark KJ;Ekker SC
转录激活因子样效应物（Transcription activator-like effector, TALEs）是一种非常有效的单分子dna靶向分子游标，用于基因座特异性基因组科学应用，包括高精度分子医学和其他基因组工程应用。tale在基因组工程中用于基因座特异性DNA编辑和成像，作为人工转录激活剂和抑制剂，以及靶向表观遗传修饰。TALENs作为核酸酶（TALEs as nucleases, TALENs）是一种有效的编辑工具，与其他自定义核酸酶系统相比，它具有高的结合特异性和更少的序列约束。广泛使用TALE的一个瓶颈是试剂的可及性。例如，一种常用的部署方法使用多管5天组装协议。在这里，我们描述了FusX，一种流线型的金门TALE组装系统，它(1)与流行的TALE主干向后兼容，(2)被功能化为单管3天的TALE组装过程，(3)只需要常用的基本分子生物学试剂，(4)具有成本效率。使用FusX已成功组装了100多个TALEN对，并在斑马鱼中对27对进行了定量测试，每对都显示出较高的体细胞和种系活性。此外，该装配系统是灵活的，与标准的分子生物学实验室工具兼容，但可以扩展与自动化实验室支持。为了演示，我们使用高度可访问且商用的液体处理机器人，使用FusX TALE工具包快速准确地组装TALEs。总之，FusX系统加速了基于tale的基因组科学应用，从基础科学筛选工作到功能基因组学测试和分子医学应用。
Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.