Targeted mutagenesis using zinc-finger nucleases in Arabidopsis
Targeted mutagenesis using zinc-finger nucleases in Arabidopsis
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DOI：
10.1073/pnas.0409339102
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发表时间：
2005-02-08
影响因子：
11.1
通讯作者：
Drews, GN
中科院分区：
文献类型：
--
作者：
Lloyd, A;Plaisier, CL;Drews, GN
定向诱变是反向遗传学的重要工具，可用于实验研究基础植物生物学或改造作物以改善重要的农业性状。尽管定向诱变在包括酵母和小鼠在内的几种模式生物中是常规的，但目前还没有有效且广泛使用的方法来在植物基因中产生定向修饰。在这项研究中，我们研究了基于锌指核酸酶（ZFN）的靶向诱变方法的功效。在此过程中，ZFN 用于在特定基因组位点产生双链断裂，随后的修复会在断裂位点产生突变。为了确定 ZFN 是否可以在高等植物基因组中的特定位点进行切割并诱导突变，我们将一种携带由热激启动子驱动的 ZFN 基因及其靶标的构建体引入拟南芥基因组中。幼苗发育过程中通过热休克诱导 ZFN 表达导致 ZFN 识别序列发生突变，每个靶标的突变频率高达 0.2 个。在 106 个 ZFN 诱导突变中，83 个（78%）是 1-52 bp 的简单缺失（中位数为 4 bp），14 个（13%）是 1-4 bp 的简单插入，9 个是伴随插入的缺失。在 10% 的诱导个体中，突变体出现在下一代中，从而证明了 ZFN 诱导突变的有效传播。这些数据表明 ZFN 可以构成植物基因定向诱变高效方法的基础。
Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. in this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 l were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFN-induced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.