Creation of intermuscular bone-free mutants in amphitriploid gibel carp by editing two duplicated runx2b homeologs

Intermuscular bones (IBs) are ubiquitous in most of freshwater farmed fishes, especially in cyprinid species, and the creation of IB-free mutants has become a hot issue in fish genetic breeding because of the potential beneficial for consumers and fish processing industry. Recently, significant progress in the mechanism underlying IB development has been achieved in zebrafish and a decisive player, runt-related transcription factor 2b (runx2b), has been identified. However, its role has not yet been validated in other farmed fishes. Amphitriploid gibel carp (Carassius gibelio) is an important aquaculture species in China, but the injuries threats with approximately 80 IBs have strongly restricted its industry development. In this study, we first identified two homeologs of runx2b (Cgrunx2b-A and Cgrunx2b-B) in gibel carp with about 91.8% identity, each of them possessing three alleles with 99.6% identities. Then, we traced the IB ossification process. Alizarin red S staining showed that gibel carp IB began to ossify at 14 approximately days post hatching (dph) and developed from the posterior to the anterior regions. Cgrunx2b-A and Cgrunx2b-B displayed similar expression pattern but differential expression level during IB development. Moreover, we applied an optimized protocol of CRISPR/Cas9 editing in the polyploid Carassius complex to disrupt the functions of CgRunx2b-A and CgRunx2b-B singly or simultaneously. IB development was unaffected by single deficiency of CgRunx2b-A or CgRunx2b-B, but simultaneous disruption of all alleles of both CgRunx2b-A and CgRunx2b-B leads to complete loss of IB. The results indicate that CgRunx2b-A and CgRunx2b-B cooperatively regulate IB development. Importantly, we created several gynogenetic gibel carp mutants, including IB-free Cgrunx2b-A−/−/−;Cgrunx2b-B−/−/− mutants and IB-less Cgrunx2b-A−/−/−;Cgrunx2b-B−/−/+ mutants. The current study not only confirms the cooperatively decisive role of two homeologs of runx2b in IB formation, but also provides a feasible gene editing-based approach to create IB-free mutants in gibel carp.