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。
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.
[1] Misra UK,Kalita J. Overview:Japanese encephalitis[J]. Prog Neurobiol,2010,91(2):108-120. DOI:10.1016/j.pneurobio. 2010.01.008.
[2] 国家卫生和计划生育委员会疾病预防控制局. 2017年全国法定传染病疫情概况[EB/OL]. (2018-02-26)[2018-03-08]. http://www.nhfpc.gov.cn/jkj/s3578/201802/de926bdb046749abb7b0a8e23d929104.shtml.
[3] Schwencke C,Braun-Dullaeus RC,Wunderlich C,et al. Caveolae and caveolin in transmembrane signaling:implications for human disease[J]. Cardiovasc Res,2006,70(1):42-49. DOI:10.1016/j.cardiores.2005.11.029.
[4] Xu QQ,Cao MM,Song HY,et al. Caveolin-1-mediated Japanese encephalitis virus entry requires a two-step regulation of actin reorganization[J]. Future Microbiol,2016,11(10):1227-1248. DOI:10.2217/fmb-2016-0002.
[5] 赵德根,王建校,刘旭,等. 小干扰RNA的研究进展[J]. 现代生物医学进展,2014,14(4):792-794. DOI:10.13241/j.cnki.pmb.2014.04.049.
[6] 陈瑶,刘青,邢微微,等. 基于CRISPR/Cas9基因编辑技术的RIP1稳定敲除细胞模型的构建及功能研究[J]. 军事医学,2017,41(2):96-100,105. DOI:10.7644/j.issn.1674-9960. 2017.02.004.
[7] Morgens DW,Deans RM,Li A,et al. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes[J]. Nat Biotechnol,2016,34(6):634-636. DOI:10.1038/nbt.3567.
[8] Zhu YZ,Xu QQ,Wu DG,et al. Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent,dynamin and caveola-mediated endocytosis pathway[J]. J Virol,2012,86(24):13407-13422. DOI:10.1128/JVI.00903-12.
[9] Wang HX,Li MQ,Lee CM,et al. CRISPR/Cas9-based genome editing for disease modeling and therapy:Challenges and opportunities for nonviral delivery[J]. Chem Rev,2017,117(15):9874-9906. DOI:10.1021/acs.chemrev.6b00799.
[10] Fu YF,Foden JA,Khayter C,et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J]. Nat Biotechnol,2013,31(9):822-826. DOI:10.1038/nbt.2623.
[11] Cong L,Ran FA,Cox D,et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science,2013,339(6121):819-823. DOI:10.1126/science.1231143.
[12] Chen JS,Dagdas YS,Kleinstiver BP,et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy[J]. Nature,2017,550(7676):407-410. DOI:10.1038/nature24268.
[13] Paquet D,Kwart D,Chen A,et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[J]. Nature,2016,533(7601):125-129. DOI:10.1038/nature17664.
[14] 卢利莎,白杨,刘鑫,等. 利用CRISPR/Cas9技术构建敲除MEIS2基因的HEK293T人胚肾细胞系[J]. 中国细胞生物学学报,2015,37(4):535-541. DOI:10.11844/cjcb.2015.04.0020.
