Shukla Vipula K, Doyon Yannick, Miller Jeffrey C, DeKelver Russell C, Moehle Erica A, Worden Sarah E, Mitchell Jon C, Arnold Nicole L, Gopalan Sunita, Meng Xiangdong, Choi Vivian M, Rock Jeremy M, Wu Ying-Ying, Katibah George E, Zhifang Gao, McCaskill David, Simpson Matthew A, Blakeslee Beth, Greenwalt Scott A, Butler Holly J, Hinkley Sarah J, Zhang Lei, Rebar Edward J, Gregory Philip D, Urnov Fyodor D
Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA.
Nature. 2009 May 21;459(7245):437-41. doi: 10.1038/nature07992. Epub 2009 Apr 29.
Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.
农业生物技术受到传统随机诱变和转基因技术低效性的限制。由于植物中靶向基因组修饰一直难以实现，植物性状工程仍然是一项费力、耗时且不可预测的工作。在此，我们报告了一种针对该问题的广泛适用且通用的解决方案：使用设计的锌指核酸酶（ZFNs），其可在目标位点诱导双链断裂。我们描述了利用ZFNs修饰作物玉米植株中的内源位点。我们表明，ZFNs的同时表达以及简单异源供体分子的导入，会在大量分离事件中导致除草剂耐受基因在预期位点精确靶向添加。经ZFN修饰的玉米植株能将这些遗传变化忠实地传递给下一代。一个目标位点IPK1的插入破坏导致了除草剂耐受性以及发育种子中肌醇磷酸谱的预期改变。ZFNs可用于任何适合DNA导入的植物物种；因此，我们的结果为基础科学和农业应用中的植物遗传操作建立了一种新策略。
