Generate a new crucian carp (Carassius auratus) strain without intermuscular bones by knocking out bmp6

Elimination of intermuscular bones (IMBs) is vital to the aquaculture industry of cyprinids. In our previous study, we characterized bmp6 as essential in the development of IMBs in zebrafish. Knockout of bmp6 results in the absence of IMBs in zebrafish without affecting growth and reproduction. Therefore, we hypothesized that bmp6 could be used to generate new cyprinid strains without IMBs by gene editing. In this study, we established a gene editing strategy for knocking out the two orthologs of bmp6 in diploid crucian carp (Carassius auratus). We obtained an F3 population with both orthologs knocked out, in which no IMBs were detected by bone staining and X-ray, indicating that the new strain without IMBs (named WUCI strain) was successfully generated. Furthermore, we extensively evaluated the performance of the new strain in growth, reproduction, nutrient components, muscle texture and structure, and metabolites in muscle. The results showed that the WUCI strain grew faster than the wild-type crucian carp at 4-month-age. The reproductive performance and flesh quality did not show significant differences between the WUCI strain and wild-type crucian carp. Moreover, the metabolomics analysis suggested that the muscle tissues of the WUCI strain significantly enriched some metabolites belonging to the Thiamine metabolism, Nicotinate and Nicotinamide Metabolism pathway, which plays beneficial effects in anti-aging, antioxidant, and anti-radiation damage. In conclusion, we established a strategy to eliminate IMBs in crucian carp and obtain a WUCI strain whose performance was expected compared to the wild-type crucian carp; meanwhile, the WUCI strain enriched some beneficial metabolites to human health in muscle tissue. This study is the first report that a cyprinid strain without IMBs, which could be stably inherited, was obtained worldwide; it provided excellent germplasm for the cyprinids aquaculture industry and a useful molecular tool for eliminating IMBs in other cyprinids.