De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks
De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks
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DOI：
10.1073/pnas.1019533108
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发表时间：
2011-02-08
影响因子：
11.1
通讯作者：
Zhu, Jian-Kang
中科院分区：
文献类型：
--
作者：
Mahfouz, Magdy M.;Li, Lixin;Zhu, Jian-Kang
位点特异性和稀有切割核酸酶是基因组工程的有价值的工具。双链DNA断裂（DSB）的产生可促进真核生物中的同源重组，并可以促进基因靶向，添加，缺失和失活。锌指核酸酶已用于生成DSB，然后用于基因组编辑，但效率低下。 III型效应子（Tales）的转录激活剂家族包含一个串联重复的中心域，可以设计以结合特定的DNA靶标。在这里，我们报告了具有用户选择的DNA结合特异性的基于HAX3的混合故事核酸酶的生成。我们表明，工程的故事核酸酶可以在体外与其目标序列结合，并且同二聚体的故事核酸酶可以在体外裂解双链DNA，如果DNA结合位点具有适当的间距和方向。烟叶中的瞬时表达测定表明，混合核酸酶在其目标序列中产生DSB，随后通过非同源性最终连接修复来修复。综上所述，我们的数据表明，基于工程故事的杂种核酸酶的可行性能够生成位点特异性DSB，以及一般而言的植物和真核生物中现场特异性基因组修饰的巨大潜力。
Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.