Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.

Characterization of ddx4 and dnd Homologs in Snakeskin Gourami (Trichopodus pectoralis) and Their Expression Levels during Larval Development and in Gonads of Males and Females.

The purpose of this study was to clone and characterize ddx4 and dnd1 homologs in snakeskin gourami (Trichopodus pectoralis) and to determine their expression levels during larval development and in the gonads of males and females. Both cDNAs contained predicted regions that shared consensus motifs with the ddx4 family in teleosts and the dnd family in vertebrates. Phylogenetic tree construction analysis confirmed that these two genes were clustered in the families of teleosts. Both ddx4 and dnd1 mRNAs were detectable only in the gonads, particularly in germ cells. These two genes were expressed during early larval development. The expression of ddx4 was high during early larval development and decreased with increasing developmental age, whereas dnd1 expression increased with developmental age. In adult fish, the expression levels of both genes were higher in the ovary than in the testis. Overall, these findings provide valuable molecular information on ddx4 and dnd, and can be applied in future reproductive biological studies relating to sex dimorphism in snakeskin gourami.