CRISPR/Cas9-mediated knock-in of masu salmon ( Oncorhyncus masou ) elongase gene in the melanocortin-4 ( mc4r ) coding region of channel catfish ( Ictalurus punctatus ) genome

Channel catfish, Ictalurus punctatus , have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou , elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor ( mc4r ) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch ( p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively ( p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls ( p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.