Exploring the Use of Alternative Promoters for Enhanced Transgene and sgRNA Expression in Atlantic Salmon Cells

This study facilitates design of expression vectors and lentivirus tools for gene editing of Atlantic salmon. We have characterized widely used heterologous promoters and novel endogenous promoters in Atlantic salmon cells. We used qPCR to evaluate the activity of several U6 promoters for sgRNA expression, including human U6 (hU6), tilapia U6 (tU6), mouse U6 (mU6), zebrafish U6 (zU6), Atlantic salmon U6 (sU6), medaka U6 (medU6), and fugu U6 (fU6) promoters. We also evaluated several polymerase type II (pol II) promoters by luciferase assay. Our results showed that hU6 and tU6 promoters were the most active among all the tested U6 promoters, and heterologous promoters (CMV, hEF1α core) had higher activity compared to endogenous Atlantic salmon promoters sHSP8, sNUC3L, sEF1α. Among endogenous pol II promoters, sEF1α and sHSP8 displayed higher activity than sNUC3L, sHSP703, sHSP7C, sXRCC1L, and sETF. We observed that extending the promoter sequence to include the region up to the start codon (ATG) resulted in a significant increase in expression efficiency for sNUC3L and sEF1α. We also show that mutating the PRDM1 motif will significantly decrease the activity of the sEF1α promoter. The presence of the PRDM1 motif in sHSP8 promoter was also associated with relatively high expression compared to the promoters that naturally lacked this motif, such as sNUC3L. We speculate that this short sequence might be included in other promoters to further enhance the promoter activity, but further experiments are needed to confirm this. Our findings provide valuable insights into the activity of different promoters in Atlantic salmon cells and can be used to facilitate further transgenic studies and improve the efficiency of transgene expression in Atlantic salmon.