Crustacean nuclear localization signals help facilitating the delivery of DNA into Australian red-claw crayfish cells

Crustacean aquaculture is rapidly expanding, and demands are supported through the application of novel technologies. One such application is the oral administration of foreign DNA, which expresses genes of interest to promote growth, health and superior quality traits. To date, there has not been any report of a viable method for large-scaled oral delivery of foreign DNA into crustaceans, although these methodologies have expanded in utilization across many plant and animal species. DNA delivery requires a thorough understanding of the transcription machinery of the host species to which it is administered. This knowledge is still unavailable in crustaceans, where the mechanism to enable nuclear entry is obscure. In this study we utilized a bioinformatics approach to shortlist Nuclear Localisation Signals (NLSs), and designed crustacean-specific NLSs to enhance the delivery of DNA into the nucleus via active transport. The NLS-plasmid complex, carrying GFP and IAG genes was transfected into Australian redclaw crayfish (Cherax quadricarinatus) primary cultured cells, resulting in protein expression of the introduced genes. These NLSs also increased the transfection efficiency in mammalian cells from 0.6% to 9% in suboptimal conditions. Hence, this integrated approach of bioinformatics and in vitro studies could help in developing a novel biotechnology to enable the large-scaled oral DNA delivery in the crustacean aquaculture industry.