基于CRISPR/Cas9系统Ⅰ型干扰素受体亚单位1基因敲除的Caco-2细胞系的构建  

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system

作　　者：刘欣奕 安妮 章青[2] 王宏[2] 孔翔羽[2] 王铭月 庞立丽[2] 段招军[2] LIU Xin-yi;AN ni;ZHANG Qing;WANG Hong;KONG Xiang-yu;WANG Ming-yue;PANG Li-li;DUAN Zhao-jun(不详;School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China)

机构地区：[1]甘肃中医药大学公共卫生学院,甘肃兰州730000 [2]中国疾病预防控制中心病毒病预防控制所国家卫生健康委员会医学病毒和病毒病重点实验室,北京102206 [3]中国医学科学院医药生物技术研究所,北京100050

出　　处：《中国生物制品学杂志》2023年第2期145-150,157,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2021YFC2301000)。

摘　　要：目的利用成簇规律间隔短回文重复序列(clustered regularly interspaced short palinmic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)系统在人结肠腺癌细胞Caco-2中敲除Ⅰ型干扰素受体亚单位1(interferon alpha/beta receptor subunit 1,IFNAR1)基因,构建IFNAR1基因敲除Caco-2细胞系。方法利用CRISPR/Cas9技术设计特异性识别IFNAR1基因外显子区的sgRNA(single guide RNA)序列,构建LentiCRISPRv2-IFNAR1-sgRNA重组质粒,慢病毒包装后感染Caco-2细胞,嘌呤霉素抗性筛选,有限稀释法培养单克隆细胞系。通过靶基因测序和Western blot法验证IFNAR1基因敲除情况;通过加入外源性IFNβ检测IFNAR1基因敲除细胞CXC趋化因子配体10(CXC chemokine ligand 10,CXCL10)和干扰素刺激基因20(interferon-stimulatd gene 20,ISG20)mRNA水平。结果质粒LentiCRISPRv2-IFNAR1-sgRNA测序结果显示插入位置均位于BsmBⅠ酶切黏性末端。共获得2株IFNAR1基因敲除单克隆细胞株,测序结果显示Caco-2-IFNAR1-KO1的IFNAR1第6个外显子发生5 bp缺失,Caco-2-IFNAR1-KO2的第7个外显子发生18 bp缺失,同时有1 bp增加。与野生型Caco-2细胞相比,Caco-2-IFNAR1-KO1和Caco-2-IFNAR1-KO2细胞IFNAR1蛋白未见表达。相比于0 ng/mL IFNβ,在50 ng/mL外源IFNβ刺激下,Caco-2-IFNAR1-KO1和Caco-2-IFNAR1-KO2细胞CXCL10基因mRNA水平(t分别为0.566和1.268,P>0.05)和ISG20基因mRNA水平(t分别为1.522和1.733,P>0.05)均无明显升高;与野生型Caco-2细胞相比,在50 ng/mL外源IFNβ刺激下,Caco-2-IFNAR1-KO1和Caco-2-IFNAR1-KO2细胞CXCL10基因mRNA水平(t分别为6.763和6.777,P<0.05)和ISG20基因mRNA水平(t分别为5.664和5.653,P<0.05)均显著降低。结论利用CRISPR/Cas9技术成功获得了IFNAR1基因敲除的Caco-2细胞株,该细胞系依赖Ⅰ型IFN受体(interferon alpha/beta receptor,IFNAR)激活的下游分子被明显抑制,为进一步探讨病毒感染Caco-2细胞后的天然免疫反应及复制包装机制提供了有力的工具。Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1)gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβstimulation,the mRNA levels of CXCL10 gene(t=0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t=1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβshowed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t=6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t=5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 kno

关 键 词：人结肠腺癌细胞 CRISPR/Cas9系统 Ⅰ型干扰素受体亚单位1基因 基因编辑 干扰素Β CXC趋化因子配体10 干扰素刺激基因20 

分 类 号：Q784[生物学—分子生物学]