Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology
Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology
复制标题
利用CRISPR/Cas9技术快速操作猪流行性腹泻病毒基因组
DOI：
10.1016/j.jviromet.2019.113772
复制
发表时间：
2020-02-01
影响因子：
3.1
通讯作者：
Xiao, Shaobo
中科院分区：
文献类型：
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作者：
Peng, Qi;Fang, Liurong;Xiao, Shaobo
猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）是一种引起哺乳仔猪腹泻的高致病性肠道冠状病毒。反向遗传学是研究病毒基因功能和产生候选疫苗的有价值的工具。本研究将PEDV强毒株AJ1102的全长感染性cDNA克隆组装到细菌人工染色体（BAC）中。拯救的病毒（rAJ 1102）在体外表现出与野生型AJ1102相似的增殖特征。利用CRISPR/Cas9技术，成功制备了将ORF 3基因替换为EGFP基因的重组病毒rAJ 1102-Delta ORF 3-EGFP，并将其增殖特性与亲本rAJ 1102进行了比较。更重要的是，使用该方法仅用一周就构建了重组PEDV rAJ 1102-Delta ORF 3-EGFP，为PEDV基因组操作提供了更有效的平台，该平台也可应用于其他RNA病毒。
Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-Delta ORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-Delta ORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.