Targeted mutagenesis of Δ5 and Δ6 fatty acyl desaturases induce multiplex-mutagenesis and lipogenesis in Atlantic salmon (Salmo salar)

Abstract With declining wild fish populations, farmed Atlantic salmon ( Salmo salar ) has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA) including 20:5n-3 and 22:6n-3. However, the introduction of plant-based oil in fish diets has reduced the content of these beneficial LC-HUFA. The capability of biosynthesis of LC-HUFAs depends on fatty acids supplied in diets and the genetic potential residing in the fish. Key proteins involved in LC-HUFA synthesis in salmon include fatty acid desaturases 2 (Fads2). In a recent study we used CRISPR/Cas9 to generate two F0 mutant strains of salmon, 1) Δ6abc/5 Mt with mutations in Δ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-c genes, and 2) Δ6bc Mt with mutations in Δ6fads2-b and Δ6fads2-c genes. The CRISPR mutated salmon (crispants) had reduced levels of LC-HUFA and expression of targeted fads2 genes. In present study we apply whole transcriptome analysis on these fads2 crispants. Our purpose is to evaluate the genetic mosaicism in fads2 crispants and the effect these mutations had on other lipid metabolism pathways in fish. Both Δ6abc/5 Mt and Δ6bc Mt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. Skipping of a CRISPR-targeted exon was observed in Δ6fads2-a gene of Δ6abc/5 Mt salmon. The Δ6abc/5 Mt fish also displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes as well as genes involved in fatty acid de-novo synthesis, fatty acid β -oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of Δ6bc Mt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability.