Investigating the application potential of TLR8a in disease-resistant breeding of <italic>Ctenopharyngodon idella</italic> via CRISPR/Cas9

The hemorrhagic disease of Ctenopharyngodon idella represents a significant constraint on the sustainable development of freshwater aquaculture. Utilizing CRISPR/Cas9 genome editing technology to screen immune negative regulatory genes and develop disease-resistant strains has emerged as a pivotal strategy in contemporary disease control. C. idella TLR8a, as a negative regulator of antiviral immunity, was targeted to assess its potential for breeding. In this study, we established F0 generation tlr8a-deficient heterozygous models to explore their immunoregulatory mechanisms in antiviral infections. The results revealed that tlr8a-knockout C. idella exhibited attenuated hemorrhagic disease symptoms, accompanied by reduced pathological lesions in both the hepatopancreas and intestine. Furthermore, there was a significant up-regulation in the expression of antiviral genes (inf1, mx2) and pro-inflammatory factors (nf-κb2, il-6). In addition, the transcription of the viral capsid protein VP56 was down-regulated following Type II grass carp reovirus infection. Additionally, peripheral blood leukocytes from tlr8a-knockout C. idella demonstrated enhanced chemotactic activity, along with increased reactive oxygen species production capacity and acid phosphatase activity. These findings suggest that TLR8a regulates antiviral innate immunity by modulating interferon signaling pathways and cytokine production, while also mediating excessive inflammatory activation. Our findings not only enhance the understanding of immunoregulatory homeostasis within TLR signaling in teleosts but also suggest a potential molecular target for developing disease-resistant fish.