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

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);Western 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。

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 positions.The editing efficiency was validated by the sanger sequencing and T-vector cloning.The changes of PCSK9 mRNA and protein expression levels were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting.Results:The results of the sanger sequeucing and T-vector cloning showed that the editing efficiencies 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-CGBENG: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.