| CRISPR/Cas13b系统对猪流行性腹泻病毒增殖的抑制作用 |
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| 引用本文： | 梅彦,杨化强,吴珍芳.CRISPR/Cas13b系统对猪流行性腹泻病毒增殖的抑制作用[J].华南农业大学学报,2019,40(6):1-7. |
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| 作者姓名： | 梅彦 杨化强 吴珍芳 |
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| 作者单位： | 华南农业大学 动物科学学院,广东 广州,510642;华南农业大学 动物科学学院,广东 广州 510642;温氏研究院,广东 云浮 527400 |
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| 基金项目： | 广东省科技计划项目（2017B020201009） |
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| 摘 要： | 【目的】猪流行性腹泻是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的一种对养猪业造成巨大损失的高致死率的传染性疫病。CRISPR/Cas13b系统精准切割和编辑RNA的功能提供了一种靶向抑制RNA病毒的策略。本研究尝试利用CRISPR/Cas13b的RNA干扰功能对PEDV的基因组RNA进行切割,以探索一种新型的PEDV病毒抑制策略。【方法】设计了4对靶向PEDV基因组不同区域的CRISPR RNA(crRNA)位点,构建了CRISPR/Cas13b打靶载体,以打靶载体转染Vero细胞,并利用PEDV感染转染细胞,检测PEDV在CRISPR/Cas13b转染细胞内的增殖情况。【结果】CRISPR/Cas13b系统对PEDV在Vero细胞的增殖具有明显的抑制作用。打靶载体U6-crRNA3和U6-crRNA4转染组相较于正常细胞组,病毒免疫荧光试验中荧光团明显减少;定量PCR结果显示,打靶载体转染组在细胞水平抑制50%以上病毒增殖量。【结论】本研究构建的CRISPR/Cas13b系统能有效抑制PEDV增殖,为开发有效的RNA病毒防控手段、建立抗病动物模型提供了新的研究策略。
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| 关 键 词： | 猪 猪流行性腹泻病毒 CRISPR/Cas13b系统 打靶质粒 RNA病毒 基因编辑 |
| 收稿时间： | 2018/11/14 0:00:00 |
Interference effect of CRISPR/Cas13b on porcine epidemic diarrhea virus |
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| MEI Yan,YANG Huaqiang and WU Zhenfang.Interference effect of CRISPR/Cas13b on porcine epidemic diarrhea virus[J].Journal of South China Agricultural University,2019,40(6):1-7. |
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| Authors: | MEI Yan YANG Huaqiang and WU Zhenfang |
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| Institution: | College of Animal Science, South China Agricultural University, Guangzhou 510642, China,College of Animal Science, South China Agricultural University, Guangzhou 510642, China;Wens'' Institute, Yunfu 527400, China and College of Animal Science, South China Agricultural University, Guangzhou 510642, China;Wens'' Institute, Yunfu 527400, China |
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| Abstract: | Objective Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious viral disease and results in high mortality of pigs and huge lost of pig industry. The CRISPR/Cas13b system can mediate a highly efficient cleavage or editing to target RNA, thereby offering a novel strategy for interfering the infection of RNA viruses. We here tried to use the CRISPR/Cas13b system to cleave the PEDV RNA genome, in order to explore a novel strategy to inhibit PEDV infection.Method We designed four CRISPR RNA (crRNA) sequences which respectively recognize four regions in the PEDV genome. The CRISPR/Cas13b targeting vectors were constructed and transfected into Vero cells. The transfected cells were infected by PEDV, and then we analyzed the viral load of PEDV in cultured cells.Result The CRISPR/Cas13b system significantly inhibited PEDV propagation in Vero cells. In the viral immunofluorescence assay, the transfected cells with targeting vectors U6-crRNA3 and U6-crRNA4 had obviously fewer fluorophores compared with normal cells. The quantitative PCR results showed that CRISPR/Cas13b decreased PEDV load in cultured cells by above 50%.Conclusion The constructed CRISPR/Cas13b system can effectively interfere the propagation of PEDV. This study provides an alternative approach for effective RNA virus prevention and control, and creation of disease-resistant pig models. |
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| Keywords: | pig porcine epidemic diarrhea virus CRISPR/Cas13b system targeting plasmid RNA virus gene editing |
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