Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development
Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development
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DOI：
10.1371/journal.pone.0223765
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发表时间：
2019-10-23
期刊：
影响因子：
3.7
通讯作者：
Pappu, Hanu R.
中科院分区：
文献类型：
--
作者：
Roy, Anirban;Zhai, Ying;Pappu, Hanu R.
白蛉传播的begomovirus对许多经济上重要的粮食、饲料和纤维作物造成严重损害。许多蔬菜作物受到严重影响，辣椒卷曲叶病毒（ChiLCV）是印度次大陆辣椒（Capsicum annuum）中最主要和广泛分布的begomvirus。最近，CRISPR-Cas9技术被用作减少双病毒在感染植物中的复制的手段。然而，这种方法被证明有一定的局限性，如逃逸突变体的进化。在这项研究中，我们使用了一种新的、基于多重向导RNA （gRNA）的CRISPR-Cas9方法，同时靶向病毒基因组的两个或多个位点。这种策略在消除ChiLCV基因组而不复发功能性逃逸突变体方面是有效的。从ChiLCV基因组中设计了6个独立的gRNA间隔序列，并在体外实验中证实了这些间隔序列的切割行为。基于上述间隔序列的组合，开发了多路gRNA表达克隆。共构建了9双工和2 - 3双工CRISPR-Cas9。测试了这些结构对烟叶中ChiLCV感染的抑制效果。结果表明，所有构建物都显著减少了病毒DNA的积累。其中，gRNA5+4、gRNA5+2和gRNA1+2三种结构体在降低病毒滴度和症状方面最为有效。T7E1检测和目标病毒基因组测序未检测到任何逃逸突变体。这种多重基因组编辑技术可能是一种有效的方法，可以触发对begemovirus的高水平抗性。据我们所知，这是第一个证明基于多重grna的植物病毒基因组编辑在减少和消除逃逸突变形成方面的有效性的报告。
Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.