Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology
Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology
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DOI：
10.1111/mpp.12375
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发表时间：
2016-09-01
影响因子：
4.9
通讯作者：
Gal-On, Amit
中科院分区：
文献类型：
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作者：
Chandrasekaran, Jeyabharathy;Brumin, Marina;Gal-On, Amit
CRISPR/Cas9（成簇规则间隔短回文重复序列）技术的发展极大地促进了植物基因组编辑。除了经典育种所提供的特性之外，这种强大的工具还可以显着改善植物性状。在这里，我们利用 Cas9/亚基因组 RNA (sgRNA) 技术破坏隐性 eIF4E（真核翻译起始因子 4E）基因的功能，展示了黄瓜 (Cucumis sativus L.) 病毒抗性的发展。 Cas9/sgRNA 构建体靶向 eIF4E 基因的 N 和 C 末端。在转化的 T1 代黄瓜植物的 eIF4E 基因靶向位点中观察到小缺失和单核苷酸多态性 (SNP)，但在推定的脱靶位点中未观察到。选择非转基因杂合eif4e突变体植物用于产生非转基因纯合T3代植物。靶向两个 eif4e 位点的 Cas9/sgRNA 后的纯合 T3 后代表现出对黄瓜脉黄化病毒（Ipomovirus）感染的免疫力以及对马铃薯病毒、西葫芦黄花叶病毒和木瓜环斑花叶病毒 -W 的抵抗力。相反，杂合突变体和非突变植物对这些病毒高度敏感。黄瓜首次通过非转基因方式培养出病毒抗性，不会明显影响植物发育，也无需长期回交，通过一项有望适用于多种作物的新技术。
Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N and C termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.