Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

TLDR

Introgression of farmed salmon escapees threatens the genetic integrity of wild stocks, and producing germ cell‑free fish may mitigate this problem. The study aimed to determine whether CRISPR‑Cas9 knockout of dnd can generate germ cell‑free Atlantic salmon in the F0 generation. sgRNA targeting alb was co‑delivered as a visual tracer, with alb knockout causing complete loss of pigmentation to identify double‑allelic mutants without mosaicism. The dnd knockout produced germ cell‑free, alb‑mutant fish, confirming alb as an effective tracer, marking the first report of dnd loss in fish and demonstrating that somatic sex differentiation can occur without germ cells, although ovarian somatic organization is compromised.

Abstract

Abstract Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd , a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer ( alb ) and the target ( dnd ) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.