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

Abstract Channel catfish, Ictalurus punctatus , have limited ability to synthesize Ω-3 fatty acids due to a lack of elongases and desaturases. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou , elongase gene driven by the common carp, Cyprinus carpio , beta-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% with 69% homozygosity/bi-allelism, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish simultaneously mutated with the ccβA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch. Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and MC4R mutants were 121.6% and 94.1% higher than in controls, respectively. Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA+DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 fish than controls. To our knowledge this is the first example of multiplexed genetic engineering and gene editing of a commercially important aquaculture species for valuable performance traits. With a high mutation rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 channel catfish appears beneficial for application on commercial farms.