Aquaculture 2022

February 28 - March 4, 2022

San Diego, California

BIOTECHNOLOGY IN AQUACULTURE: A CRUSTACEAN OOCYTES DELIVERY TOOL FOR GENE SILENCING AND EDITING

Isam Khalaila*, Shany Cohen and Omer Iluz

Avram and Stella Goldstein–Goren Department of Biotechnology Engineering, and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel

 



Oviparous animals are characterized by an elaborated yolk production process and packaging in the oocytes before egg-laying. The major yolk protein (vitellin) is usually produced as vitellogenin outside the egg (Vg) and internalized into oocytes by receptor-mediated endocytosis (RME). Like many other crustacean species, M. rosenbergii vitellogenin is expressed in the hepatopancreas. M. rosenbergii vitellogenin possesses 2537 amino acids and shares at least 33% identity with other decapod crustacean vitellogenin such as shrimps, crabs, and crayfishes. Vg contains several domains, among them the lipid-binding domain that is involved in yolk-lipid vesicles formulation.  Upon arrival to the oocytes, the Vg-receptor (VgR) extracellular domain interacts with a distinct amino acid sequence of the Vg and internalizes it to form yolk droplets. One distinctive characteristic of the VgR family is their role in massive internalization and accumulation of lipoproteins.  Vitellogenesis is recognized by an immense accumulation of the Vg in the oocyte that will serve the embryo’s metabolic needs for development and growth. For that reason, we predicted that Vg endocytosis could be used as a valuable tool for oligonucleotides’ high throughput delivery into the oocyte. Indeed, a specific Vg-derived peptide sequence (Vg24) was found capable of oocytes’ specific entry by in vitro and in vivo means. However, a peptide with the same amino acid composition but scrambled order (scVg24) could not enter the oocytes. Vg24 synthesized with nine Lysine-Histidine repeats or recombinantly expressed downstream of a double-strand RNA binding domain (dsRBP) were conjugated successfully with dsRNA by electrostatic or affinity interactions, respectively. The KH-Vg24 and the dsRBP-Vg24 were able to in vitro piggyback PAX6 dsRNA into M. rosenbergii oocyte. The piggybacked PAX6 dsRNA led to eye development retardation in embryos of a treated mother. Alignment analysis of the M. rosenbergii Vg24 shared 85% identity with the corresponding Vg peptide sequences from other decapod species. The peptide similarity proposes cross-reactivity between the M. rosenbergii peptide and the VgR of other decapod species. The developed tool might serve to deliver other than dsRNA molecules into crustacean oocytes, which may prove to be a powerful high throughput tool for functional genomics investigation in crustacean embryos, immunity, and transgenic animal production.