Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly
Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly
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DOI：
10.1101/gr.089417.108
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发表时间：
2009-07-01
期刊：
影响因子：
7
通讯作者：
Kim, Jin-Soo
中科院分区：
文献类型：
--
作者：
Kim, Hye Joo;Lee, Hyung Joo;Kim, Jin-Soo
锌指核酸酶（ZFN）技术在研究、医学和生物技术中的广泛应用受到缺乏一种方便、快速和公开的方法来合成功能性ZFN的阻碍。在这里，我们描述了一种高效且易于实践的模块化组装方法，使用公开可用的锌指来制造可以修饰人类细胞中预定基因组位点DNA序列的ZFNs。我们合成并测试了数百个ZFNs，以针对人类CCR5基因（HIV感染所需的共同受体）的数十个不同位点，并发现许多这些核酸酶在CCR5序列中诱导了位点特异性突变。由于携带CCR5零突变的人类细胞是功能性的和正常的，这些ZFNs可能用于(1)敲除CCR5以产生抵抗艾滋病患者HIV感染的t细胞，或(2)在基因治疗应用的“安全位点”插入治疗基因。
Broad applications of zinc finger nuclease (ZFN) technology-which allows targeted genome editing-in research, medicine, and biotechnology are hampered by the lack of a convenient, rapid, and publicly available method for the synthesis of functional ZFNs. Here we describe an efficient and easy-to-practice modular-assembly method using publicly available zinc fingers to make ZFNs that can modify the DNA sequences of predetermined genomic sites in human cells. We synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene-a co-receptor required for HIV infection-and found that many of these nucleases induced site-specific mutations in the CCR5 sequence. Because human cells that harbor CCR5 null mutations are functional and normal, these ZFNs might be used for (1) knocking out CCR5 to produce T-cells that are resistant to HIV infection in AIDS patients or (2) inserting therapeutic genes at "safe sites'' in gene therapy applications.