DF-1细胞ANP32A基因缺失株的基因编辑及其对AIV复制的抑制效应OA北大核心CSTPCD

Avian influenza virus(AIV)can hijack the host ANP32A protein to bind to influenza virus polymerase and promote AIV replication.However,the molecular mechanism by which chANP32A regulates influenza virus replication is still unknown.In this study,we designed and synthesised sgRNA sequences targeting amino acid(AA)129 of chANP32A using adenovirus-mediated CRISPR/Cas9 gene editing,constructed Ad-SpCas9 adenoviral vector,and packaged adenovirus.The recombinant adenovirus was used to infect DF-1 cells through subclonal culture,PCR amplification and genome sequencing,and three cell lines with deletion of 6,21 and 24 bases were identified.Cells were infected with H9N2 avian influenza virus and measured by RT-qPCR,TCID50,and influenza virus polymerase activity methods,RT-qPCR results showed that the viral genome RNA copy number of the KO-6,KO-21,and KO-24 cell lines was significantly reduced,the viral growth curves were measured by TCID50,and the viral titres were significantly reduced,the in-fluenza virus dual luciferase reporter.The results showed that the viral polymerase activity of influenza virus in the deletion cell lines was reduced by 30-3 000 times.It was found that KO-6 and KO-21 cell lines inhibited AIV replication,KO-24 cell line significantly inhibited AIV replication,and the deletion of chANP32A 125-132 AA even more significantly inhibited AIV replication.This study lays the foundation for the study of chANP32A gene function and the development of gene-edited chickens with resistance to avian influenza viruses and no change in the biological function of chANP32A.