Generation of blunt snout bream without intermuscular bones by runx2b gene mutation

Blunt snout bream (Megalobrama amblycephala) is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones (IBs). Our previous study proved that runx2b is the key gene for the IBs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without IBs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of IBs. In order to further obtain the blunt snout bream without IBs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without IBs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 (1 insertion, 2 deletion), 13 (8 insertion, 5 deletion) and 7 (1 insertion, 6 deletion) mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that IBs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish (P > 0.05). RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type (with IBs) and runx2b mutant F1 individuals (without IBs) at 120 dpf. The results showed that the basic muscle nutritional composition (moisture, crude protein, crude lipid and crude ash), and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage (P > 0.05). This study proves that the runx2b gene has an essential role for IBs formation in aquaculture species and lays an important foundation for obtaining a new blunt snout bream strain without IBs, which provides a good example for the application of IBs-free characters from model fish to commercial fish.