锌指核酸酶(ZFN)由锌指蛋白(ZFP)结构域和Fok I核酸内切酶的切割结构域人工融合而成,是近年来发展起来的一种可用于基因组定点改造的分子工具。ZFN可识别并结合特定的DNA序列,并通过切割这一序列的特定位点造成DNA的双链断裂(DSB)。在此基础上,人们可以对基因组的特定位点进行各种遗传操作,包括基因打靶、基因定点插入、基因修复等,从而能够方便快捷地对基因组实现靶向遗传修饰。这种新的基因组定点修饰方法的突出优势是适用性好,对物种没有选择性,并且可以在细胞和个体水平进行遗传操作。文章综述了ZFN技术的研究进展及应用前景,重点介绍ZFN的结构与作用机制、现有的靶点评估及锌指蛋白库的构建与筛选方法、基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,为开展这一领域的研究工作提供参考。
Zinc finger nuclease(ZFN) is an artificially engineered hybrid protein that contains a zinc finger protein(ZFP) domain and a Fok I endonuclease cleavage domain.It has recently emerged as a powerful molecular tool for targeted ge-nome modifications.ZFNs recognize and bind to specific DNA sequences to generate a double-strand break(DSB) by its nuclease activity.Based on this finding,various genetic methods,including gene targeting(gene disruption),gene addition,gene correction etc.,are being designed to manipulate the genomes of different species at specific loci.One particular ad-vantage of this new technique is its broad applications,which can be employed to generate desirable inheritable mutations both at the organismal level and at the cellular level.Here,we review the recent progress and prospects of ZFN technology.This article focused on the mechanism of how it works,currently available target assessment,ZFP library construction and screening methods,target modification strategies,as well as a collection of specie and genes that have been successfully modified by ZFN.This review will provide a useful reference for researchers who are interested in applying this new tech-nique in their studies.