CRISPR/Cas9介导的RPSA基因敲除细胞系的建立及其应用

【目的】利用CRISPR/Cas9技术建立RPSA基因缺失的乳仓鼠肾细胞(baby hamster kidney cells,BHK21)细胞系,为开展RPSA调控病毒复制机制研究提供工具;同时,初步探究RPSA对塞内卡病毒复制的影响。【方法】根据GenBank中仓鼠的RPSA基因序列找到产生不同转录本的共同外显子段,设计并合成4对引导RNA(sgRNA),分别构建至PX330载体中;经过筛选选择打靶活性较高的PX330-RPSA-sgRNA2质粒用于后续敲除细胞系构建。将PX330-RPSA-sgRNA2质粒转染BHK21细胞后,通过有限稀释法筛选单克隆细胞,通过Westernblot及序列测定检测RPSA基因的敲除。通过Westernblot及qPCR分析比较塞内卡病毒在野生型及RPSA基因敲除BHK21细胞中的复制差异。【结果】Western blot检测及序列测序证实了RPSA基因敲除单克隆细胞构建成功。进一步研究发现,塞内卡病毒在RPSA基因敲除细胞中的复制水平明显低于其在野生型BHK21细胞中复制的水平。【结论】成功构建了RPSA基因敲除的BHK21细胞系,首次表明RPSA对塞内卡病毒的复制具有重要作用,为进一步开展RPSA在细胞内调控病毒复制机制研究奠定基础。

Construction of RPSA gene knockout BHK21 cell line using the CRISPR/Cas9 system

Objective] Generating ribosomal protein SA (RPSA) knockout baby hamster syrian kidney (BHK21) cells using the clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology, and evaluation of the regulatory effect of RPSA on Senecavirus A (SVA) replication. Methods] We designed 4 pairs of guide RNAs (sgRNA). After screening, the PX330-RPSA-sgRNA2 recombinant plasmid was used for construction of RPSA gene knockout cell line. PX330-RPSA-sgRNA2 was transfected into BHK21 cells, and the monoclonal cell was screened by limited dilution method. The knockout of RPSA was evaluated by Western blotting. The replicative difference of SVA in the wildtype and RPSA knockout cells was investigated by Western blotting and qPCR analysis.Results] The Western blotting and Sanger sequencing results showed that RPSA within the BHK21 cell line was knocked out. We further investigated the replication of SVA in the RPSA knockout cells which showed that SVA replication was dramatically decreased in RPSA knockout cells comparing with that in the wildtype cells. Conclusion] The RPSA gene knockout BHK21 cell line was successfully constructed, and RPSA played an important role during SVA replication. The constructed cell line will be a useful tool for exploiting RPSA functions.