A library of TAL effector nucleases spanning the human genome
A library of TAL effector nucleases spanning the human genome
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DOI：
10.1038/nbt.2517
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发表时间：
2013-03-01
影响因子：
46.9
通讯作者：
Kim, Jin-Soo
中科院分区：
文献类型：
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作者：
Kim, Yongsub;Kweon, Jiyeon;Kim, Jin-Soo
转录激活因子样（TAL）效应物核酸酶（TALEN）可以容易地被工程化以结合特定的基因组基因座，从而能够引入精确的遗传修饰，例如基因敲除和添加。在这里，我们提出了一个基因组规模的TALEN集合，用于在人类细胞中进行有效和可扩展的基因靶向。我们选择了在基因组中其他地方没有高度相似序列的靶位点，以避免脱靶突变，并使用高通量Golden-Gate克隆系统组装了18，740个蛋白质编码基因的TALEN质粒。一项涉及124个基因的初步试验表明，所有TALEN都是活跃的，并以高频率破坏其靶基因，尽管其中两个TALEN只有在使用DNA甲基转移酶抑制剂使其靶位点部分去甲基化后才变得活跃。我们使用我们的TALEN文库产生单基因和双基因敲除细胞，其中NF-κ B信号通路被破坏。与用短干扰RNA处理的细胞相比，这些细胞显示出明确的信号转导抑制。
Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-kappa B signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.