Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon

Abstract

Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.

Surrogate broodstock to enhance biotechnology research and applications in aquaculture

“…This approach has successfully produced sterile embryos in various species including zebrafish ( Saito et al, 2008 ), masu salmon, rainbow trout ( Yoshizaki et al, 2016 ), Atlantic salmon ( Wargelius et al, 2016 ), sterlet ( A. ruthenus ; Linhartová et al, 2015 ), grass puffer ( Yoshikawa et al, 2020 ) and Chinese rosy bitterling ( Octavera and Yoshizaki, 2019 ). Furthermore, germ cells in dnd1 KO salmon could be rescued by co-injecting the salmon dnd1 mRNA together with Cas9/gRNA targeting dnd1 ( Güralp et al, 2020 ). This strategy would allow fertile surrogate broodstocks that produce sterile offspring.…”

A refinement to gene editing in Atlantic salmon using asymmetrical oligonucleotide donors

“…DNA was extracted from caudal fins using DNeasy Blood & Tissue kit (Qiagen). DNA extracted from the fin has previously been shown to be broadly representative for the whole fish 5,25 . A fragment covering the entire CRISPR target sites for slc45a2 and dnd1 was amplified with a two-step fusion PCR (as described in Gagnon et.al 2014) to prepare for Illumina MiSeq.…”

“…Moreover, this could also be an advantage for aquaculture species in general (e.g trout, sea bass, tilapia). This technology offers an exciting opportunity to insert traits of interest into the recently demonstrated fertile but genetically sterile salmon 25 . This fish will produce sterile offspring and may therefore represent the future salmon aquaculture by combining sterility and other favorable traits induced by HDR, such as disease resistance.…”

“…The genetic progress in selective breeding programs is also limited by the heritability of the target traits, and the standing genetic variation in the broodstock. NBT using CRISPR/Cas9-induced HDR can offer new solutions and opportunities in these areas 2,25 .…”

A comparison of triploid and diploid Atlantic salmon (Salmo salar) performance and welfare under commercial farming conditions in Norway

“…The farming of sterile salmon has been proposed as a possible solution to minimize the impacts on wild populations and thus a way to make the salmon aquaculture industry more sustainable (Aarset et al 2020;Benfey 2016). Although there exist several different sterilization techniques (Güralp et al 2020), to date, the only commercially available method of producing sterile salmon is to triploidise newly fertilized eggs (Golpour et al 2016).…”