应用C-to-G碱基编辑器对PCSK9基因靶向敲除的研究
Research on the targeted knockout of the PCSK9 gene by C-to-G basic group editor
刘惠聪 1曹小芳 2万子睿 3安莉莎 2金孝华 2马旭1
作者信息
- 1. 国家卫生健康委科学技术研究所(北京,100081);北京协和医学院研究生院
- 2. 国家卫生健康委科学技术研究所(北京,100081)
- 3. 国家卫生健康委科学技术研究所(北京,100081);首都医科大学附属北京朝阳医院
- 折叠
摘要
目的:使用C-to-G碱基编辑器(CGBE)Td-CGBE-NG靶向敲除huh-7肝癌细胞系前蛋白转化酶枯草溶菌素9(PCSK9)基因,以CBE(AncBE4max)为参照,评价Td-CGBE-NG对该基因的敲除效果.方法:使用引入终止密码子的策略敲除huh-7肝癌细胞系PCSK9基因.构建碱基编辑器Td-CGBE-NG和AncBE4max,构建重组质粒sg386和sg555,将 Td-CGBE-NG 和 sg386,AncBE4max 和 sg555 分别共转染 huh-7 细胞,实现 c.1447C＞G 和 c.1953C＞T 的转换,在两位点引入终止密码子S386X(TGA)和Q555X(TAG),使用sanger测序和T载体克隆验证编辑效率;实时荧光定量聚合酶链反应(RT-qPCR)和蛋白免疫印迹法(Western blot)检测PCSK9基因mRNA和蛋白表达水平的变化.结果:sanger测序和T载体克隆结果显示,Td-CGBE-NG和AncBE4max的编辑效率分别为c.1447C＞G 33％和c.1953C＞T 25％;RT-qPCR 结果显示,Td-CGBE-NG 使 PCSK9 mRNA 的表达量降低(t=17.29,P＜0.0001);West-ern blot结果表明,两组huh-7细胞蛋白表达量均明显下降(AncBE4max:t=5.57,P＜0.01;Td-CGBE-NG:t=4.912,P＜0.01).结论:Td-CGBE-NG可以有效敲除huh-7肝癌细胞系的PCSK9基因,抑制该基因mRNA和蛋白的表达,为后续Td-CGBE-NG的应用奠定基础.
Abstract
Objective:To evaluate the efficacy of the C-to-G basic group editor(CGBE)Td-CGBE-NG in targeted knockout of the proprotein convertase subtilisin/kexin type 9(PCSK9)genes of huh-7 hepatocellular carcinoma cells based on the reference of CBE(AncBE4max).Methods:The PCSK9 gene of the huh-7 hepatocellular carcinoma cells was knocked out by a strategy involving the introduction of a stop codon.The basic group editors of Td-CGBE-NG and AncBE4max were constructed,and the recombinant plasmids of sg386 and sg555 were generated.Co-transfection of Td-CGBE-NG and sg386,or AncBE4max and sg555,was conducted to the huh-7 cells to induce the conversion of c.1447C＞G and c.1953C＞T,thereby introducing the stop codons S386X(TGA)and Q555X(TAG)at the two posi-tions.The editing efficiency was validated by the sanger sequencing and T-vector cloning.The changes of PCSK9 mR-NA and protein expression levels were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Results:The results of the sanger sequencing and T-vector cloning showed that the editing efficien-cies of Td-CGBE-NG and AncBE4max were c.1447C＞G 33％and c.1953C＞T 25％.RT-qPCR analysis revealed the reduction of PCSK9 mRNA expression by Td-CGBE-NG(t=17.29,P＜0.0001).Western blotting confirmed protein expression levels in both groups of huh-7 cells had decreased significantly(AncBE4max:t=5.57,P＜0.01;Td-CGBE-NG:t=4.912,P＜0.01).Conclusion:Td-CGBE-NG can knockout the PCSK9 gene of the huh-7 hepatocellular carcinoma cells effectively,which leading to the inhibition of the gene mRNA and protein expressions.These findings lay a foundation for the future application of Td-CGBE-NG.
关键词
前蛋白转化酶枯草溶菌素9(PCSK9)/CRISPR/Cas/碱基编辑/huh-7肝癌细胞系Key words
Proprotein convertase subtilisin/kexin type 9/CRISPR/Cas/Basic group editing/Huh-7 Hepatocellular carcinoma cell line引用本文复制引用
基金项目
国家重点研发项目(2016YFC1000307)
中央级公益性科研院所基本科研业务费专项资金(2022GJM08)
出版年
2024
NETL
NSTL
万方数据