Kandavelou Karthikeyan, Chandrasegaran Srinivasan
Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Methods Mol Biol. 2009;544:617-36. doi: 10.1007/978-1-59745-483-4_40.
Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.
锌指核酸酶（ZFNs）是经过定制设计的分子剪刀，可被设计用于在特定DNA序列处进行切割。ZFNs将锌指蛋白（ZFPs）与FokI限制酶的非特异性切割结构域结合在一起。ZFNs的DNA结合特异性可以通过实验轻松改变。对ZFN识别特异性的这种轻松操控使得人们能够将靶向双链断裂（DSB）导入基因组。靶向DSB通过同源重组（HR）在该特定切割位点将局部基因靶向刺激几个数量级。因此，ZFNs已成为一种重要的实验工具，不仅可对植物和哺乳动物的基因组，还可对许多其他生物的基因组进行位点特异性和永久性改变。定制ZFNs的工程涉及许多步骤。第一步是在基因组内选定的染色体靶标处或其附近识别一个ZFN位点，ZFNs将与之结合并切割。第二步是设计和/或选择各种将以高特异性和亲和力与选定靶位点结合的ZFP组合。然后使用寡核苷酸通过聚合酶链反应（PCR）组装所设计的ZFPs的DNA编码序列。第三步是将ZFP构建体与FokI切割结构域融合。然后使用兔网织红细胞体外转录/翻译系统将ZFNs表达为蛋白质，并对蛋白质产物进行DNA切割特异性检测。
