On genome editing in embryos and cells of the freshwater prawn Macrobrachium rosenbergii

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) technology provides the means for accurate genomic editing. It has been applied in many kinds of cells and animals for functional genomic studies and for precise selective breeding. Nonetheless, this method has not yet been applied in one of the most important – and well studied – decapod crustacean aquaculture species, the giant freshwater prawn Macrobrachium rosenbergii. We thus established two CRISPR platforms for M. rosenbergii—the first through direct injection into early-stage embryos (entire organism genome editing) and the second by electroporation of a primary embryonic cell culture. The systems were calibrated by optimizing Cas9 concentrations, delivery methods and editing efficiencies. Editing patterns utilizing multiple guides were examined through next generation sequencing. Our results showed a wide range of editing efficiencies in embryos, in some cases reaching as high as 100%. In contrast, in primary embryonic cell cultures, the highest editing efficiency obtained reached a maximum of 64%. In addition, there was a striking difference between the two platforms in terms of the pattern of deletions around the Cas9 cut site. This finding suggests distinct repair mechanisms in the two systems, which calls for further clarification. A phenotypic proof of concept was provided through the investigation of an early acting paired box protein 6 (Pax6) transcription factor, which showed clear effects on eye development in edited embryos and larvae. The current study lays down the foundations for precise functional genomic research and applications of genome editing in crustacean species for both aquaculture and sustainable biocontrol, opening opportunities for the creation of selected crustacean lines with distinct attributes.