CRISPR/Cas9-mediated editing of Δ5 and Δ6 desaturases impairs Δ8-desaturation and docosahexaenoic acid synthesis in Atlantic salmon (Salmo salar L.)

The in vivo functions of Atlantic salmon fatty acyl desaturases (fads2), Delta 6fads2-a, Delta 6fads2-b, Delta 6fads2-c and Delta 5fads2 in long chain polyunsaturated fatty acid (LC-PUFA) synthesis in salmon and fish in general remains to be elucidated. Here, we investigate in vivo functions and in vivo functional redundancy of salmon fads2 using two CRISPR-mediated partial knockout salmon, Delta 6abc/5(Mt) with mutations in Delta 6fads2-a, Delta 6fads2-b, Delta 6fads2-c and Delta 5fads2, and Delta 6bc(Mt) with mutations in Delta 6fads2-b and Delta 6fads2-c. F0 fish displaying high degree of gene editing (50-100%) were fed low LC-PUFA and high LC-PUFA diets, the former containing reduced levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids but higher content of linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids, and the latter containing high levels of 20:5n-3 and 22:6n-3 but reduced compositions of 18:2n-6 and 18:3n-3. The Delta 6abc/5(Mt) showed reduced 22:6n-3 levels and accumulated Delta 6-desaturation substrates (18:2n-6, 18:3n-3) and Delta 5-desaturation substrate (20:4n-3), demonstrating impaired 22:6n-3 synthesis compared to wildtypes (WT). Delta 6bc(Mt) showed no effect on Delta 6-desaturation compared to WT, suggesting Delta 6 Fads2-a as having the predominant Delta 6-desaturation activity in salmon, at least in the tissues analyzed. Both Delta 6abc/5(Mt) and Delta 6bc(Mt) demonstrated significant accumulation of Delta 8-desaturation substrates (20:2n-6, 20:3n-3) when fed low LC-PUFA diet. Additionally, Delta 6abc/5(Mt) demonstrated significant upregulation of the lipogenic transcription regulator, sterol regulatory element binding protein-1 (srebp-1) in liver and pyloric caeca under reduced dietary LC-PUFA. Our data suggest a combined effect of endogenous LC-PUFA synthesis and dietary LC-PUFA levels on srebp-1 expression which ultimately affects LC-PUFA synthesis in salmon. Our data also suggest Delta 8-desaturation activities for salmon Delta 6 Fads2 enzymes.