Estabilishment Of TALEN, CRISPR-Cas9 And ENU-mutagenesis In Common Carp (Cyprinus Carpio) And Their Applications On Osteoblast And Muscle Fiber Development

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. Common carp also serves as a vertebrate model for extensive studies in ecology and evolution, environmental toxicology, physiology, nutrition, immunology, development, breeding and transgenics.However, its intermuscular bones prevent it from being a favorable delicacy in some regions of the world. Hence genetic modifications aiming to improve its qualities and economic value as well as to facilitate its utilities as a biological model are highly demanded. But 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, bmp2 a, spp1, opg, and muscle suppressor gene mstn. Phylogenetic analysis showed that common carp have two sp7 genes and four mstn genes, while zebrafish have only one sp7 gene and two mstn genes, consistent with tetraploidy nature of common carp genome.TALEN were shown to induce mutations with high effciencies in the target coding sites of sp7, runx2, spp1 and mstn. With CRISPR-Cas9, the two common carp sp7 genes, sp7 a and sp7 b, were mutated individually, all resulting in severe bone defects. Micro-CT analysis showed the craniofacial bones and centrum bones of these sp7a- and sp7b-mutated carps develop slowly than those of control groups. However, sp7a-CRISPR mutated carps showed more obvious bone defects than sp7b-CRISPR mutated carps. Alizarin Red staining showed that sp7a-CRISPR common carps exhibit conspicuous bone defects including opercula and maxilla insufficiency, the irregular hemal spines and deformed centrums, and shorter inter-muscular bones than uninjected wild-type control. On the other hand, mstnba mutated carps have grown significantly more muscle cells and larger muscle fibers with heavier body weight than uninjected carps. Moreover, the body weight exhibits a phenotype-genotype correlation that the higher the mutation rate, the heavier the fish. Western blotting results showed that CRISPR-Cas9-induced mutations impair the Mstn signal pathway of the skeletal muscle of common carp. qRT-PCR and H&E staining both showed mstnba-CRISPR mutated F0 carps display hyperplasia as well as hypertrophy.We also employed CRISPR-Cas9 to generate double mutant carps(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.Moreover, as an effective approach for genetic improvement of agriculture, ENU was used to treat mature sperms of common carp, and then ENU-treated sperms fertilized wild-type eggs. Results indicated that high concentration of ENU caused morphological abnormalities of embryos at segmentation stage. The ENU-treated F1 population showed large variations in growth and nearly 16% of them showed obvious bone defects during adult stage. These results demonstrate that ENU treatment of mature sperms is a potentially useful approach for generation of mutated common carps.