Targeting and editing the second exon of bmp6 gene results in a silver carp with reduced intramuscular bones

Intermuscular bones (IBs) are needle-like bones frequently observed within the muscle septa of cyprinid fish, which adversely affects fish palatability and economic value. The deletion of bmp6 has been shown to lead the absence of IBs in fish, without affecting normal growth. Therefore, we hypothesised that knockout of bmp6 via gene editing could product the IBs-free silver carp ( Hypophthalmichthys molitrix ). In this study, CRISPR/Cas9 technology was successfully used to target and disrupt exon 2 and exon 4 of bmp6 in silver carp, respectively, resulting in the generation of two bmp6 -deficient chimeras, named chimera-E2 (CH-E2) and chimera-E4 (CH-E4). Both two types of chimeras are capable of normal growth and development; nonetheless, their IBs exhibit distinct defective phenotypes. Specifically, both CH-E2 and CH-E4 groups exhibit bending of the IBs; however, 30 % of the samples in the CH-E2 group showed a reduction in IBs, which is not observed in the CH-E4 group. Subsequent transcriptome sequencing revealed that DEGs identified both in CH-E2 and CH-E4 groups were significant enrichment in “HIF-1 signaling pathway”, with upregulated of egf and down-regulated of vegfa , which are hypothesized to contribute to the bending of IBs. DEGs identified only in CH-E2 group were significant enrichment in “osteoclast differentiation”, “NF-kappa B signaling pathway” and “cytokine-cytokine receptor interactions”, indicating a specificity unique to CH-E2 group. This was associated with a marked upregulation of fosb and tnf , which promote osteoclast development and inhibit osteoblast differentiation, consequently leading to a reduced number of IBs in CH-E2 group. In conclusion, we investigated the impact of bmp6 deficiency on the development of IBs in silver carp and elucidated its potential molecular mechanisms. The findings offer a reliable knockout sites on exon 2 of the bmp6 gene, providing valuable insights for the future breeding of silver carp new varieties devoid of IBs. • CRISPR/Cas9 technology was employed to create the generation of two bmp6 -deficient chimeras. • Both chimeras developed normally with curved IBs; CH-E2 had a 30% reduction in IBs, while CH-E4 showed no significant change. • In bmp6-deficient silver carp, egf expression increased and vegfa expression decreased, possibly causing IBs to bend. • The CH-E2 group showed higher fosb and tnf expression, promoting osteoclast growth, possibly reducing IBs.