Abstract: Objective To establish a stable CAV-1 gene knockout cell line by CRISPR/Cas9 system in neuroblastoma cell line (SH-SY5Y), and to investigate its function on Japanese encephalitis virus (JEV) infection. Methods The sgRNA sequences targeting EXON of human CAV-1 gene were designed according to the principles of CRISPR/Cas9, and then were cloned into PX459 plasmid.The SH-SY5Y cells transfected with the recombinant plasmids were selected by puromycin to gain the CAV-1 knockout cells. The cells with CAV-1 knockout effect were verified by Western blotting. The differences of JEV infection on CAV-1 knockout cells and wild type cells were detected by immunofluorescence. Results The recombinant plasmids were verified by enzyme cutting and gene sequencing and were successfully constructed. The protein of CAV-1 was undetected in SH-SY5Y cells after transfection and screening of monoclonal cell by puromycin and the CAV-1 knockout SH-SY5Y cells were successfully constructed. Compared to the wild type cells, the infectivity of JEV on the CAV-1 knockout cells decreased by 90% (P<0.001). Conclusion The CAV-1 gene was knocked out successfully by CRISPR/Cas9 system in SH-SY5Y cells. This cell line can be used to further study the mechanism of JEV infection.

Key words: Gene editing, Caveolin, Neuron cells, Japanese encephalitis virus

摘要： 目的 利用CRISPR/Cas9基因编辑系统建立小窝蛋白1（CAV-1）基因敲除的神经母细胞瘤细胞株（SH-SY5Y），并验证其对流行性乙型脑炎病毒（JEV）的易感性。方法 根据CRISPR/Cas9靶点设计原则，基于人源CAV-1基因的外显子区（EXON）序列设计小向导RNA（sgRNA），将sgRNA连接到载体PX459质粒上。将重组质粒转染至SH-SY5Y细胞中，使用嘌呤霉素筛选稳定敲除CAV-1蛋白的SH-SY5Y细胞，并用免疫印迹法验证CAV-1蛋白的敲除效果。免疫荧光法比较CAV-1基因敲除的SH-SY5Y株与野生型细胞株对JEV感染性的差异。结果 经酶切和测序鉴定，重组真核表达质粒构建正确，转染并筛选的单克隆细胞中没有CAV-1蛋白的表达，成功构建CAV-1基因敲除的SH-SY5Y细胞株。与野生型SH-SY5Y细胞株相比，CAV-1基因敲除的细胞株对JEV的感染性下降约90%（P<0.001）。结论 利用CRISPR/Cas9系统成功构建CAV-1基因敲除的SH-SY5Y细胞株，该细胞株可用于进一步研究CAV-1蛋白在JEV感染中的作用机制。

关键词: 基因编辑, 小窝蛋白, 神经细胞, 流行性乙型脑炎病毒