CRISPR/Cas9-mediated resistance to cauliflower mosaic virus
CRISPR/Cas9-mediated resistance to cauliflower mosaic virus
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DOI：
10.1002/pld3.47
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发表时间：
2018-03-01
期刊：
影响因子：
3
通讯作者：
Wang, Xiaofeng
中科院分区：
文献类型：
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作者：
Liu, Haijie;Soyars, Cara L.;Wang, Xiaofeng
病毒性疾病是全球农作物产量损失的主要原因。在优良作物品种中选育抗病基因（R基因）已成为标准和最具成本效益的做法。然而，R基因介导的耐药性受到遗传资源中可用R基因的限制，在许多情况下，受菌株特异性的限制。因此，开发新的广谱抗病毒策略具有重要意义。CRISPR-Cas9 （clustered regularly interspaced palindromic repeat, CRISPR-associated）编辑系统已被用于赋予对人类病毒和几种植物单链DNA双病毒的抗性，指出了CRISPR-Cas9系统在病毒控制方面的可能应用。在这里，我们证明了病毒对花菜花叶病毒（CaMV）的强抗性，CaMV是一种具有双链DNA基因组的副逆转录病毒，可以通过cas9介导的病毒外壳蛋白序列的多重靶向实现。我们进一步表明，小干扰rna （siRNA）被产生，并且大多数映射到单导rna （sgRNA）的30端，尽管非常低水平的siRNA也映射到间隔区。然而，这些sirna并不是抑制CaMV感染的原因，因为如果Cas9不存在，就不会产生耐药性。我们还在一些转基因植物系统感染的叶片中观察到编辑过的病毒，在外壳蛋白编码序列的sgRNA靶位点上出现与cas9诱导的DNA断裂一致的短缺失或插入。这些被编辑的外壳蛋白，在大多数情况下，导致早期翻译停止，从而导致无功能外壳蛋白。我们还在这些受感染的转基因植物中恢复了野生型CP序列，表明这些编辑过的病毒基因组被野生型外壳蛋白包装。我们的数据表明，CRISPR-Cas9系统可以通过进一步的修饰用于植物副逆转录病毒的病毒控制。
Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost-effective practice. However, R gene-mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad-spectrum antiviral strategies. The CRISPR-Cas9 (clustered regularly interspaced palindromic repeat, CRISPR-associated) editing system has been employed to confer resistance to human viruses and several plant single-stranded DNA geminiviruses, pointing out the possible application of the CRISPR-Cas9 system for virus control. Here, we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double-stranded DNA genome, can be achieved through Cas9-mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 30 end of single-guide RNAs (sgRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9-induced DNA breaks at the sgRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, nonfunctional coat proteins. We also recovered wild-type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild-type coat proteins. Our data demonstrate that the CRISPR-Cas9 system can be used for virus control against plant pararetroviruses with further modifications.