Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Viruses. 2022 May 24;14(6):1119. doi: 10.3390/v14061119.
nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV and genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target and , which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV and genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.
核型多角体病毒（BmNPV）是一种导致家蚕严重疾病的病原体。在之前的研究中，我们证明了通过使用 CRISPR/Cas9 系统破坏 BmNPV 的 和 基因，转基因家蚕对 BmNPV 感染表现出抗性。在这里，我们使用相同的策略同时靶向 和 ，这对于 BmNPV 的复制是必不可少的。PCR 检测证实，在表达小向导 RNA（sgRNA）和 Cas9 的感染 BmNPV 的转基因家蚕中，病毒 DNA 在靶向序列处诱导了双链断裂。生物测定和 qPCR 显示，与对照组相比，BmNPV 的复制和死亡率在转基因家蚕中显著降低。显微镜观察显示，在感染 BmNPV 的野生型家蚕中，中肠细胞降解，但在转基因家蚕中没有。这些结果表明，使用 CRISPR/Cas9 系统破坏 BmNPV 的 和 基因的转基因家蚕可以成功预防 BmNPV 感染。我们的研究不仅为 CRISPR 抗病毒系统提供了更多的替代靶标，而且旨在为病毒感染研究和昆虫防治的应用提供新的思路。
