Optimizing RNA Delivery Strategies for Gilthead Seabream (Sparus aurata) SaB-1 Cells

Aquaculture is the world’s fastest-growing food production sector, yet it faces major challenges, including limited understanding of the biology of farmed species. In this context, cell lines are becoming increasingly relevant for studying complex traits and their underlying genetic basis. The in vitro application of molecular tools—such as RNA interference and genome editing—can help disentangle the molecular basis of target traits. However, the lack of protocols for in vitro manipulation is limiting progress in the field. Here, we optimized RNA transfection protocols in SaB-1 cells derived from gilthead seabream, one of the most important farmed species in Mediterranean aquaculture. Despite its importance, in vitro research on this species has been scarce to date. Our results show that RNA delivery in this cell line can be achieved using either electroporation or FUGENE®-SI-based chemical transfection, although electroporation resulted in lower survival (around 35% versus 76% for chemical transfection). Finally, as a proof-of-concept, both delivery strategies were tested for CRISPR/Cas9-mediated gfap gene editing, which was achieved with efficiencies of around 10%. This study provides useful methodological advances for functional genomics in aquaculture, offering RNA delivery that enables genome-editing strategies and can accelerate molecular research and trait improvement in gilthead seabream.