Characterization of dead-end knockout Atlantic salmon offspring: mutation frequency, gonad phenotype and exploring the possibilities for using these as surrogates in germ cell transplantations

Abstract

Reproductively sterile fish in aquaculture has received considerable attention in recent years and is interesting for several reasons. Using sterile Atlantic salmon (Salmo salar) in fish farming could contribute to solving the challenges with genetic introgression into wild strains, as well as the fish welfare and economic problems related to early sexual maturation. One approach to produce sterile salmon is by targeted mutagenesis of the germ cell specific dead-end (dnd) gene, using the gene editing technique CRISPR-Cas9. dnd knockout (KO) is shown to induce sterility in several species. A sterile, dnd KO salmon model produced at the Institute of Marine Research, named VIRGIN, is in addition injected with dnd mRNA to allow the formation of germ cells despite its genetic sterility. Subsequently, it is expected that offspring from these genetically sterile gametes are sterile, which would represent an efficient way to mass-produce this model. In this thesis, dnd mutation rates and corresponding gonad phenotypes in VIRGIN offspring (F1 generation) from three different crosses were characterized. As expected, all double-allelic dnd mutants (dnd-/-) were germ cell free (GCF). Surprisingly, CGF fish were also found among heterozygous dnd+/- mutants in two of the crosses, and even among fish with a wild-type (WT) genotype (dnd+/+) in one cross. Another method to mass-produce sterile fish, is by surrogacy; transplanting germ cells that carry any mutation causing sterility, into surrogates that will mature and spawn these donor-derived gametes. Germ cell transplantations can be performed in or between species and can be used for additional purposes like production of mono-sex populations and species conservation. It is beneficial that the surrogates are sterile, to avoid competition between donor-derived and endogenous germ cells within the gonad. To investigate if VIRGIN salmon are suitable as surrogates for transplanted germ cells, WT spermatogonia was transplanted into newly hatched VIRGIN F1 larvae. Although studies have been conducted with germ cell transplantations in salmonids, no previous studies have demonstrated a successful germ cell transplantation in a 100 % sterile Atlantic salmon. After 7 months, expression levels of germ cell markers dnd, vasa and piwil1 in the gonads from surrogates were measured to get an impression of whether the transplantations were successful or not. Interestingly, elevated expression levels of dnd, vasa and piwil1 was found in one dnd-/- fish, which is a strong indication that the transplantations have worked, and that sterile dnd KO salmon can be used for germ cell transplantations. However, further research is needed to confirm 100 % that the germ cells are donor-derived and not endogenous.