Gene targeting in plants: 25 years later
Gene targeting in plants: 25 years later
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DOI：
10.1387/ijdb.130194hp
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发表时间：
2013-01-01
影响因子：
0.7
通讯作者：
Fauser, Friedrich
中科院分区：
文献类型：
--
作者：
Puchta, Holger;Fauser, Friedrich
在植物转基因研究开始仅5年后，首次在烟草中实现了基因打靶（GT）。不幸的是，与随机整合相比，通过同源重组（HR）的靶向整合的频率如此之低，以至于GT不能在高等植物中建立为可行的技术。又花了25年的时间和巨大的努力来开发克服这一挑战所需的知识和工具，至少对一些植物物种来说是这样。在某些情况下，参与HR的蛋白质的过表达或阴性选择标记的使用在一定程度上改善了GT。1996年开发了一种有效的解决方案，当时使用序列特异性核酸内切酶在靶基因座处诱导双链断裂（DSB）。因此，GT频率显著提高。此后，主要限制是缺乏在基因组特定位点诱导DSB所需的工具。随着锌指核酸酶（ZFN）的发展，这些工具变得可用，并且在2005年取得了突破，当时ZFN被用于靶向烟草中的标记基因。随后，在玉米、烟草和拟南芥中靶向内源基因座。最近，我们的基因工程工具箱已经扩展，增加了更多类型的位点特异性核酸内切酶，大范围核酸酶，转录激活因子样效应核酸酶（TALEN）和CRISPR/Cas系统。我们认为，有针对性的基因组修饰将成为常规在不久的将来在作物植物中使用这些核酸酶沿着与新开发的在植物GT技术。
Only five years after the initiation of transgenic research in plants, gene targeting (GT) was achieved for the first time in tobacco. Unfortunately, the frequency of targeted integration via homologous recombination (HR) was so low in comparison to random integration that GT could not be established as a feasible technique in higher plants. It took another 25 years and great effort to develop the knowledge and tools necessary to overcome this challenge, at least for some plant species. In some cases, the overexpression of proteins involved in HR or the use of negative selectable markers improved GT to a certain extent. An effective solution to this problem was developed in 1996, when a sequence-specific endonuclease was used to induce a double-strand break (DSB) at the target locus. Thus, GT frequencies were enhanced dramatically. Thereafter, the main limitation was the absence of tools needed to induce DSBs at specific sites in the genome. Such tools became available with the development of zinc finger nucleases (ZFNs), and a breakthrough was achieved in 2005 when ZFNs were used to target a marker gene in tobacco. Subsequently, endogenous loci were targeted in maize, tobacco and Arabidopsis. Recently, our toolbox for genetic engineering has expanded with the addition of more types of site-specific endonucleases, meganucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system. We assume that targeted genome modifications will become routine in the near future in crop plants using these nucleases along with the newly developed In planta GT technique.