Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
Mol Biol Rep. 2020 May;47(5):3369-3376. doi: 10.1007/s11033-020-05409-3. Epub 2020 Apr 15.
Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.
植物病毒是全球农业产业中导致重大经济损失的最主要因素。因此，由于病毒的性质不断变化，每年都有许多研究致力于培育抗病毒作物品种。最近，基因组工程方法已被用于对真核病毒产生干扰。特别是 CRISPR-Cas9 基因组编辑技术的研究结果，为培育抗病毒作物提供了一种可行的解决方案。在这项研究中，我们探索了利用 CRISPR-Cas9 获得抗 TYLCV 番茄品种的可能性。此外，为了克服 Cas9 的任何潜在脱靶效应，我们使用诱导启动子在病毒攻击时启动 Cas9 活性。Cas9 载体通过 rgsCaM 启动子转化，rgsCaM 启动子是 RNAi 的内源性沉默子，在病毒攻击后过表达。采用 Golden Gate 克隆方法构建 sgRNAs。设计 sgRNAs 时使用了 TYLCV 的内含子区和外壳蛋白编码序列。通过实时 PCR 分析，与对照植物相比，渗透敏感的 Money Maker 品种的病毒 DNA 显著减少或积累延迟。该结果表明，在 CRISPR-Cas9 构建中使用诱导启动子的有效性。
