Targeted genome editing in pluripotent stem cells using zinc-finger nucleases
Targeted genome editing in pluripotent stem cells using zinc-finger nucleases
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DOI：
10.1016/j.ymeth.2010.12.019
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发表时间：
2011-04-01
期刊：
影响因子：
4.8
通讯作者：
Cathomen, Toni
中科院分区：
文献类型：
--
作者：
Bobis-Wozowicz, Sylwia;Osiak, Anna;Cathomen, Toni
锌指核酸酶（ZFNs）是一种人工设计的核酸酶，能够在复杂的基因组内切割预先指定的靶DNA。ZFNs由一个非特异性核酸内切酶结构域与一个工程化的DNA结合结构域融合而成，该DNA结合结构域将核酸酶活性束缚在选定的染色体位点上。核酸内切酶诱导的DNA双链断裂会触发细胞的DNA损伤反应，导致通过精确的同源重组（HR）或易错的非同源末端连接（NHEJ）对双链断裂进行修复。因此，ZFNs是在多种哺乳动物细胞类型（包括胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs））中进行靶向基因组工程的有力工具。作为多能干细胞中基因组编辑的范例，我们描述了在小鼠胚胎干细胞中使用ZFNs通过非同源末端连接在无选择的情况下产生基因敲除等位基因，或者通过采用不同选择方案的同源重组来产生基因敲除等位基因。（C）2011爱思唯尔公司。保留所有权利。
Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes. (C) 2011 Elsevier Inc. All rights reserved.