State Key Laboratory of Resource Insects, Southwest University, Chongqing 400715, China.
State Key Laboratory of Resource Insects, Southwest University, Chongqing 400715, China; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Southwest University, Chongqing 400716, China.
Int J Biol Macromol. 2024 Oct;278(Pt 2):134773. doi: 10.1016/j.ijbiomac.2024.134773. Epub 2024 Aug 14.
Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(-)/sgorf76(-), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(-)/sgorf76(-). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control.
病毒病对畜牧业和植物栽培构成重大威胁。CRISPR/Cas9 介导的病毒基因靶向编辑为抗病毒治疗提供了一种有前途的方法。家蚕是一种经济上重要的昆虫，易感染家蚕核型多角体病毒（BmNPV），病毒爆发给养蚕业造成严重的经济损失。在这里，我们鉴定出 BmNPV orf76 是一种病毒晚期基因，与 Autographa californica 多角体病毒 Ac93 高度相似。orf76 的缺失消除了 BmNPV 的增殖，并阻碍了感染性芽生病毒的产生。我们生成了一个转基因组 Cas9(+)/sgorf76(+)，它不影响家蚕的生长和发育，并且证明转基因组 Cas9(+)/sgorf76(+)在 sgorf76 位点有效地切割 orf76，导致感染后 120 小时出现大片段缺失，没有观察到脱靶效应。生存分析表明，与对照系 Cas9(-)/sgorf76(-)相比，转基因组 Cas9(+)/sgorf76(+)的家蚕具有更高的存活率，无论 BmNPV 的接种剂量如何。此外，与对照系 Cas9(-)/sgorf76(-)相比，转基因组 Cas9(+)/sgorf76(+)中 BmNPV DNA 拷贝数和病毒基因的表达水平明显受到抑制。结果为 Cas9 介导的针对 BmNPV 的抗病毒治疗提供了一个有前途的靶标，并为杆状病毒基因功能研究和鳞翅目害虫防治提供了新的见解。
