Infectious and parasitic diseases pose significant challenges to tilapia aquaculture, causing notable economic losses and adversely affecting the welfare of these fish species. Utilizing cell lines as experimental models plays a pivotal role in advancing our comprehension of infectious diseases, providing a valuable platform for studying the intricate interactions between pathogens and the host. In combination with genome editing technologies, such as CRISPR/Cas systems, it becomes possible to assess the specific functions of genes within these systems. This approach allows research into potential targets for enhancing resistance to diseases such as Tilapia lake virus (TiLV) in tilapia aquaculture. The ANP32 family, acidic (leucine-rich) nuclear phosphoprotein 32 kDa, play various endogenous roles in regulating gene expression, intracellular transport, and cell death. Interestingly, avian ANP32 proteins are also critical for avian influenza polymerase activity and can influence viral replication, a key factor in viral proliferation. In this current study, we knocked out anp32a and demonstrated a significant increase in levels of resistance to TiLV. We then analysed the transcriptomic and proteomic response of knockout cells to the virus to characterize the mechanism of resistance. This work has the potential to impact strategies for disease control in Nile tilapia.