利用CRISPR/Cas9技术建立STAT1敲除细胞系及其功能验证

Establishment and Functional Verification of A STAT1 Knockout Cell Line by Gene Editing Using CRISPR/Cas9

信号转导和转录活化因子1(Signal transducer and activator of transcription 1,STAT1)是干扰素信号通路中的关键分子,在对抗病毒和其他病原体的免疫反应中发挥重要作用,构建STAT1敲除细胞系有助于研究其在病毒感染过程中的作用与机制。本研究利用CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats-Cas9)基因编辑技术构建STAT1基因敲除细胞系。首先构建STAT1基因特异性打靶载体,后通过慢病毒转导人非小细胞肺癌细胞系(Human non-small cell lung cancer cells,A549),嘌呤霉素筛选稳定转导的细胞后进行单克隆化,最后对获取的单克隆细胞在分子水平上进行敲除及敲除回补验证,成功构建一株STAT1基因敲除(STAT1 gene knockout,STAT1-KO)的A549细胞,且该细胞系可维持正常细胞活性与增殖能力。以汉滩病毒(Hantaan virus,HTNV)感染验证,发现在该细胞系中病毒复制水平明显升高。本研究成功构建了一株STAT1-KO A549细胞,可为抗病毒药物和干扰素信号通路研究提供有力工具。

Signal transducer and activator of transcription(STAT)-1 is a key molecule in the interferon signaling pathway.It plays an important part in the immune response against viruses and other pathogens.Therefore,construction of a STAT1 knockout cell line(STAT1-KO)can aid study of its role in viral infection.STAT1-KO was constructed using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene-editing technology.First,a STAT1-specific targeting vector was constructed,and human non-small-cell lung cancer(A549)cells were transduced by lentivirus.Stable transduced cells were screened by puromycin and monoclonal antibodies.Finally,the obtained monoclonal cells were verified at the molecular level,and a STAT1-KO A549 cell line was constructed.This cell line could maintain normal cell activity and proliferation ability.Validation was undertaken with infection by the Hantaan virus.The level of viral replication was increased significantly in this cell line.In conclusion,we constructed a STAT1-KO A549 cell line,which could be a powerful tool for the study of antiviral drugs and interferon signaling pathway.