Mani Mala, Kandavelou Karthikeyan, Dy Fei Jamie, Durai Sundar, Chandrasegaran Srinivasan
Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
Biochem Biophys Res Commun. 2005 Sep 23;335(2):447-57. doi: 10.1016/j.bbrc.2005.07.089.
Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.
锌指核酸酶（ZFN）介导的基因靶向正迅速成为用于植物和哺乳动物基因组（包括人类基因组）的“基因编辑”和“定向诱变”的强大工具。ZFN介导的基因靶向为分子生物学家提供了对植物和哺乳动物基因组进行位点特异性操作和永久修饰的能力。在ZFN介导的基因靶向成为生物技术中广泛使用的高效实用工具之前，能够轻松生产ZFN并快速表征其体外序列特异性切割特性是一个先决条件。在此，我们报告了分别靶向两个小鼠基因（mTYR和mCFTR）以及两个人类基因（hCCR5和hDMPK）内特定内源序列的ZFN的设计、工程改造及快速体外表征。这些工程化的ZFN以序列特异性方式识别质粒底物中编码的各自同源DNA位点，并且正如预期的那样，它们在选定的靶位点诱导双链断裂。
