CRISPR/Cas9介导的USP30基因敲除细胞系的建立及其对塞内卡病毒增殖的影响

HEK-293T cells with USP30 knockout:construction using the CRISPR/Cas9 system and effects on Senecavirus A replication

【目的】利用规律成簇的间隔短回文重复序列/Cas9核酸酶(clustered regularly interspaced short palindromic repeats/Cas9 nuclease,CRISPR/Cas9)技术建立USP30基因敲除的人胚胎肾细胞(human embryonic kidney 293T cells,HEK-293T)细胞系,为开展宿主泛素特异性蛋白酶30(ubiquitin-specific protease 30,USP30)蛋白的功能研究建立了细胞模型;同时,初步探究USP30蛋白在病毒感染过程中的作用。【方法】根据Ensemble数据库查询USP30基因序列,定位USP30在基因组中不同转录本重叠区的第一个外显子段,设计并合成2对引导RNA(single guide RNAs,sgRNA),分别构建在pX459载体中;将pX459-USP30-sgRNA质粒转染HEK-293T细胞,并用嘌呤霉素处理,筛选出转染阳性的细胞,然后通过有限稀释法筛选单克隆细胞,通过Western blotting及测序检测USP30基因的敲除。通过Western blotting及实时荧光定量PCR分析比较塞内卡病毒(Senecavirus A,SVA)在野生型和USP30基因敲除HEK-293T细胞中的复制差异。【结果】Western blotting及测序证实USP30基因敲除单克隆细胞系构建成功。进一步实验发现,SVA在USP30基因敲除细胞中的复制水平显著低于野生型细胞。【结论】成功构建USP30基因敲除的HEK-293T细胞系,首次证明USP30对SVA的复制具有促进作用,为进一步揭示USP30相关免疫反应和SVA感染过程的作用机制提供了良好的细胞模型,也为开展宿主USP30蛋白调控病毒复制的机制研究提供了关键工具和一定的理论依据。

[Objective]The clustered regularly interspaced short palindromic repeats/Cas9 nuclease(CRISPR/Cas9)system was employed to knock out the ubiquitin-specific protease 30(USP30)gene from human embryonic kidney 293T(HEK-293T)cells,which established a cell model for studying the function of USP30 and the regulatory role of this protein in virus replication.[Methods]According to the sequence of the first exon in the overlapping region of different transcripts of USP30 in Ensemble,we designed two pairs of single guide RNAs(sgRNAs)and constructed the recombinant plasmids pX459-USP30-sgRNAs.The HEK-293T cells were transfected with pX459-USP30-sgRNA and treated with puromycin for the screening of transfection-positive cells,from which the monoclonal cells were screened out by limited dilution method.We then employed Western blotting and DNA sequencing to evaluate the knockout of USP30 and compare the replication of Senecavirus A(SVA)in the wild type and the cells with USP30 knockout.[Results]We confirmed that USP30 was knocked out from HEK-293T cells by Western blotting and DNA sequencing.Further experiments revealed that the replication level of SVA in the cells with USP30 knockout was significantly lower than that in the wild type,indicating a positive role of USP30 in SVA replication.[Conclusion]We successfully constructed the HEK-293T cells with USP30 knockout and confirmed that USP30 played a positive role in SVA replication.The constructed cell line provides a good cell model for further deciphering the mechanisms of USP30-associated immune response and SVA infection process and serves as a tool for studying the regulatory role of USP30 in viral replication.