Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.
Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.
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DOI：
10.1002/0471142727.mb1216s103
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发表时间：
2013-07
影响因子：
--
通讯作者：
Joung, J Keith
中科院分区：
文献类型：
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作者：
Reyon, Deepak;Maeder, Morgan L;Khayter, Cyd;Tsai, Shengdar Q;Foley, Jonathan E;Sander, Jeffry D;Joung, J Keith
使用转录激活因子样效应子（TALE）重复序列制备的定制DNA结合结构域作为广泛适用的研究工具的重要性正在迅速增长。TALE核酸酶（TALEN）由融合到FokI核酸酶结构域的TALE重复序列的工程化阵列组成，已成功用于多种不同生物体和细胞类型中的定向基因组编辑。TALE转录因子（TALE-TF）由连接至转录调节结构域的工程化TALE重复阵列组成，已用于上调或下调人细胞和植物中内源基因的表达。在这里，我们描述了一个详细的协议，用于实践最近描述的快速连接为基础的自动化固相高通量（FLASH）组装方法。FLASH能够使用自动化液体处理机器人或使用多通道移液器手动高通量构建工程化TALE重复序列。利用FLASH的自动化版本，单个研究者可以在一天内构建多达96个编码不同长度TALE重复序列的DNA片段，然后在一周或更短的时间内克隆这些片段以构建序列验证的TALEN或TALE-TF表达质粒。我们还在这里描述了对Zinc Finger和TALE Targeter（ZiFiT Targeter）网络服务器（http：//www.example.com）的改进，这些改进有助于高通量设计和构建FLASH TALE重复阵列。ZiFiTBeta.partners.org
Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput.