Recent biotechnological innovations have allowed the development of new approaches to apply genetic engineering to non-model organisms, including the Atlantic salmon. Key innovations include the development of next generation sequencing which allows fast and cost effective analysis of whole genomes, transcriptomes and epigenomes. Studies on gene functions became feasible for many following the introduction of the CRISPR-Cas9 methodology, which can specifically target and mutate genes in any organism, thus also allowing studies on the genetics of key traits. We have explored both methodologies with the aim to target two major problems in today's salmon aquaculture; (i) escaped fish and (ii) early maturation.

(i) Genetic introgression mediated into wild populations by farmed salmon escapees is a major concern, and is currently one of the factors limiting the expansion of the Norwegian salmon industry. To address this problem, we are investigating the possibility to induce sterility by vaccination in salmon. Exploring the testicular transcriptome of immature and maturing salmon and comparing it to other tissues enabled us to select suitable vaccine targets. We have utilized CRISPR-Cas9 technology to elucidate the function of candidate genes in the development and survival of germ cells in salmon. Due to the long generation time of salmon we have had to analyze complete loss of function in F0. To avoid analysis of mosaic individuals, we simultaneously induced CRISPR-Cas9-mediated mutations in the albino (alb) and in the target gene. We observed that complete loss of pigmentation indicated bi-allelic disruption of alb but also of the second gene that was targeted. This methodology allowed producing germ cell-free salmon, in which we could show that Atlantic salmon, like loach and goldfish, has a germ cell-independent sex determination system. We are also following growth and performance of these fish. In addition we are at this time investigating a sterility vaccine using this target.

(ii) Precocious maturity in salmon farms leads to increased susceptibility to disease and hypo-osmoregulatory problems. Also early maturity increase risk of genetic interactions with wild fish, since escaped early maturing fish are more likely to survive until maturation and spawn in the wild. Currently the problem with early maturation is partially controlled by using artificial light regimes. However, both increasing sea water temperatures and increased use of closed farming systems in the growth phase, can increase the incidence of early maturation. To unravel genetics behind early maturity at sea, we have re-sequenced pools of salmon returning to spawn after 1 or 3 sea winters in 6 rivers in Western Norway. The study revealed one major selective sweep, which covered 74 significant SNPs in a 370 kb region of chromosome 25. Genotyping domesticated fish narrowed the haplotype region to four SNPs covering the vgll3 gene, which included two missense mutations that could explain 33-36% phenotypic variation. This study demonstrates that a single locus plays a highly significant role in governing age at maturity in this species. However, the causative genetic variant(s) at this locus are unknown. We are now exploring the functionality of the vgll3 gene in salmon using CRISPR-Cas9 technology. In conclusion, the use of new biotechnological tools has enhanced significantly and will keep doing so our knowledge on the life cycle biology of salmon. This knowledge will be instrumental for refining aquaculture breeding systems.