CRISPR/Cas9介导的ALKBH5基因敲除细胞系的建立及其应用OA

This study aimed to clarify the role and mechanism of the ALKBH5 gene in foot-and-mouth disease virus(FMDV)replication through its m6A demethylase activity.A BHK-21 cell line with ALKBH5 knockout was successfully established using CRISPR/Cas9 technology,and the knockout efficiency was confirmed by Western-blot.The CCK-8 assay indicated no significant change in cell viability after knockout.Following FMDV infection of wild-type and knockout cells,it was found that ALKBH5 knockout significantly enhanced the expression of the viral 2C protein,viral RNA transcription levels,and viral titers,suggesting that ALKBH5 deletion promoted FMDV replication.Further enzymatic activity rescue experiments demonstrated that complementation with wild-type ALKBH5 suppressed viral replication,whereas the key catalytic site mutant(R131A)lost this inhibitory activity,confirming that the inhibitory effect of ALKBH5 on FMDV replication depended on its demethylase activity.This study reveals that ALKBH5 functions as a host restriction factor that negatively regulates FMDV replication through m6A demethylation,providing a theoretical basis for further elucidating the mechanisms of viral infection and developing novel antiviral drugs.