Introduction
Aquaculture is a key food production system supporting the needs of a rapidly growing global population. Despite its promise, the production efficiency and fish health and welfare is negatively impacted by infectious disease. The impact of infectious diseases can be reduced by improving prevention and treatment options, however development of more effective tools requires a deeper understanding of host-pathogen interactions. Using CRISPR-Cas9 genome editing in combination with other sequencing and molecular tools, we interrogated the function of an anti-viral ubiquitin-ligase gene; trim25, capable of immune activation. The aim was to deepen our understanding of the regulation of immune response, with the aim of implementing this knowledge for vaccination and selective breeding programs.
Results
Two copies of trim25 were identified in the Atlantic salmon genome, despite multiple other TRIM25-like annotations. Infection of an Atlantic salmon cell line (SHK-1) with infectious salmon anaemia virus (ISAV) identified unbalanced inducation of trim25 paralog expression, consistent with published results from ISAV infection in addition to POLY (I:C) stimulation, confirming the subfunctionalization of trim25 paralogs. CRISPR-Cas9 mediated knock-out of paralogous copies of trim25 confirmed the anti-viral function of Ssa02 trim25, but not Ssa12 trim25. The antiviral mechanism of trim25 was confirmed with RNA sequencing of infected knock-out cells. Lastly, over-expression of TRIM25 resulted in the reduction of viral replicaton in SHK-1 cells, confirming the anti-viral role of trim25.
Discussion
Taken together, these results confirm that trim25 is a anti-viral E3 ubiquitin ligase, which conserves the mechanism characterized in model species; namely regulation of the RIG-l like receptor pathway. Trim25 has previously been identified as a putative gene behind a QTL for resistance to ISAV in Atlantic salmon, and as such further understanding of this gene presents an opportunity to select for more resistant fish. Additionally, the toolset developed in this project offers the opportunity to interrogate gene function in Atlantic salmon.