Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common carp

Abstract

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide. However, the tetraploidy genome and long generation-time of the common carp have made its breeding and genetic studies extremely difficult. Here, TALEN and CRISPR-Cas9, two versatile genome-editing tools, are employed to target common carp bone-related genes sp7, runx2, bmp2a, spp1, opg, and muscle suppressor gene mstn. TALEN were shown to induce mutations in the target coding sites of sp7, runx2, spp1 and mstn. With CRISPR-Cas9, the two common carp sp7 genes, sp7a and sp7b, were mutated individually, all resulting in severe bone defects; while mstnba mutated fish have grown significantly more muscle cells. We also employed CRISPR-Cas9 to generate double mutant fish of sp7a;mstnba with high efficiencies in a single step. These results demonstrate that both TALEN and CRISPR-Cas9 are highly efficient tools for modifying the common carp genome, and open avenues for facilitating common carp genetic studies and breeding.

A vertebrate specific and essential role for sp7/osterix in osteogenesis revealed by gene knock-out in the teleost medaka

“…In striking difference to recent reports on osx mutants in carp and zebrafish (Kague et al, 2016;Zhong et al, 2016), we observed strong defects in embryonic and larval bone formation after osx knockout in medaka, leading to early adult lethality in homozygous mutants and vertebral malformations in heterozygous carriers (Fig. S11).…”

“…There are several possibilities to explain the incomplete phenotype of the osx mutants in carp and zebrafish. The carp has a tetraploid genome (Xu et al, 2014) and harbours two copies of sp7, both of which function in bone formation (Zhong et al, 2016). In such situations, the still-intact second copy can be expected to take over, at least partially, the function of the mutant copy.…”

“…Two recent reports described the generation of osx mutants in members of the cyprinid family, the common carp (Cyprinus carpio) and zebrafish (Danio rerio) (Kague et al, 2016;Zhong et al, 2016). Interestingly, these mutants are viable and exhibit recognizable bone defects only at later stages of life.…”

Zebrafish sp7 mutants show tooth cycling independent of attachment, eruption and poor differentiation of teeth

“…In teleost fish, the role of sp7 in bone and teeth has been recently reported (Kague et al, 2016;Niu et al;Yu et al, 2017;Zhong et al, 2016). We have described previously that zebrafish null for sp7 show severe defects in bone growth and mineralization, consistent with the roles ascribed to the gene through GWAS in humans, mouse and medaka fish.…”

Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9

“…Mutations are inheritable due to a mosaic targeting of the germline. For instance, the CRISPR/Cas9 system has been induced mutations in many animals including zebrafish (Chang et al., , Hwang et al., , Jao et al., ), common carp (Zhong et al., ), tilapia (Li et al., ), and Atlantic salmon (Fraidenraich et al., ). Among different species, an optimized codon Cas9 is usually required for editing efficiency.…”