Altering the Fatty Acid Biosynthetic Pathway with Multiple Transgenes to Elevate Omega-3 Fatty Acids Production in Channel Catfish (Ictalurus punctatus)

Abstract

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. The channel catfish (Ictalurus punctatus) is one of leading freshwater aquaculture species in the US, but it has low levels of EPA and DHA compared to some fish, such as salmon. The biosynthesis of EPA and DHA in fish requires desaturation and elongation procedures. Elongase elovl2 gene and desaturase Δ4 fad, Δ5 fad, Δ6 fad, fat-1 (Δ15) and fat-2 (Δ12) are all important genes limiting EPA and DNA biosynthesis pathway. To improve EPA and DHA content, we generated transgenic channel catfish carrying multiple elongase and desaturase transgenes, using several CRISPR/Cas9 strategies. Channel catfish carrying masu salmon (Oncorhynchus masou) elovl2 transgene were generated with random integration, CRISPR/Cas9 HDR (homology-directed repair)-mediated knockin (KI) and 2H2OP ( two-hit by gRNA and two oligos with a targeting plasmid) methods. Integration rate of these three methods were 27.3%, 19% and 37.5%. DHA content of muscle increased by 20.7% - 62% in transgenic P1 fish produced by CRISPR/Cas9 technology compared to the non-transgenic fish. To elevate KI efficacy and achieve multiple genes KIs in transgenic breeding program, we introduced a new strategy which enables transgene integration with numerous sites of genome by targeting long repeated sequence (LRS). Using this simple strategy, we successfully generated transgenic fish carrying masu salmon elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, at one generation.