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.
Proprotein convertase subtilisin/kexin type 9CRISPR/CasBasic group editingHuh-7 Hepatocellular carcinoma cell line
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