Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):12028-33. doi: 10.1073/pnas.0914991107. Epub 2010 May 27.
We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, approximately 20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFN-induced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.
我们在此报告了一种通过锌指核酸酶（ZFN）的调控表达来靶向突变拟南芥基因的有效方法，该酶可在特定靶标位点产生 DNA 双链断裂。通过 Oligomerized Pool ENgineering（OPEN）——一个公开的、基于选择的平台，制造了识别拟南芥 ADH1 和 TT4 基因的 ZFN，该平台产生高质量的锌指阵列。ADH1 和 TT4 ZFN 受雌激素诱导启动子的控制，并通过花浸转化引入拟南芥植物。诱导表达 ADH1 或 TT4 ZFN 的初级转基因拟南芥幼苗的体细胞突变频率分别为 7%或 16%。诱导的突变通常是插入或缺失（1-142 bp），定位于 ZFN 切割位点，可能源自非同源末端连接对染色体断裂的不精确修复。对于表达 ADH1 ZFN 的初级转基因植物中的 69%和表达 TT4 ZFN 的转基因植物中的 33%，突变被传递到下一代。此外，约 20%的产生突变的植物在 ADH1 或 TT4 上是纯合突变的，这表明两个等位基因都被破坏了。选择 ADH1 和 TT4 作为本研究的靶标是因为它们具有可选择或可筛选的表型（adh1，烯丙醇抗性；tt4，种皮中缺乏花青素）。然而，观察到的 ZFN 诱导突变的高频率表明，可以通过简单地对初级转基因植物的后代进行 PCR 和 DNA 测序筛选，很容易恢复靶向突变。总之，我们的结果表明，现在应该可以获得任何拟南芥靶基因的突变，而不管其突变表型如何。
