Establishment of a CRISPR/Cas9-Based Genome Editing System in Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii is a globally farmed decapod species of high economic and nutritional value, but its genetic improvement has been constrained by the lack of an efficient genome editing workflow. Here, we optimized a CRISPR/Cas9 microinjection system for targeted mutagenesis in M. rosenbergii embryos. A controlled mating-based approach enabled routine collection of one-cell embryos, and developmental staging identified 0.5-2 h post-fertilization as the optimal injection window. Optimization of the needle tip (~1 μm) minimized mechanical damage, lowering early embryo mortality to ~10%. Using this system, the eye-development gene MrPAX6 and the sex-related gene MrIAG were successfully edited. MrPAX6 editing produced a 46.9% mutation rate and an average 50% reduction in eye pigment area. MrIAG editing achieved an 84% mutation rate, while post-injection survival to hatching was approximately 35%. These results establish an efficient and reproducible CRISPR/Cas9 editing system in M. rosenbergii, providing a foundation for functional genomics and genetic improvement in this commercially important prawn species.