Disruption of mstna and mstnb gene through CRISPR/Cas9 leads to elevated muscle mass in blunt snout bream (Megalobrama amblycephala)

Abstract

Myostatin (mstn) belongs to the TGF-β family, which is mainly involved in the negative regulation of myogenesis in vertebrates. Upon implementation of several gene-editing techniques (ZFN, TALEN, RNAi and CRISPR/Cas9), disruption of mstn gene locus in several animal species has been shown to result in elevated muscle growth. As there are numerous reports on gene editing of mstn in animals (common carp, yellow catfish, channel catfish, etc.), we aimed to study the complete disruption of mstn in economy-farmed fish. Here, we analyzed and characterized mstna and mstnb in blunt snout bream. In blunt snout bream, both mstna and mstnb peptides were found to possess unique structural characteristics of the TGF-β family. We designed three target sites (targets 1, 2, and 3) to generate knock out mutants within the mature mRNA sequence of mstna or mstnb that were located in exon 1, exon 3, and exon 3, respectively. The fertilized eggs (1–2 cell stage) were injected with 1 nL mixture of gRNA (100 pg) and Cas9 protein (900 pg). The mutation efficiency of mstna targets 1, 2, and 3 was 5.1%, 8.2%, and 10.0% with mutation types 5, 4, and 12, respectively. Similarly, the mutation efficiency of mstnb targets 1, 2, and 3 was 15.3%, 14.7%, and 26.0% with mutation types 2, 3, and 5, respectively. At the age of six months, the body weight, height, and thickness of both mstna- (mstna+/−) and mstnb- (mstnb+/−) deficient fish were found to be increased significantly (p < .01) when compared to those of wild-type control fish. Moreover, the average muscle fiber sizes of both mstna+/− and mstnb+/− fish were significantly larger (p < .01) than that of control fish. Our results provide valuable data associated with the regulation of muscle development and growth in blunt snout bream. These results also provide a basis and reference for the application of CRISPR/Cas9 (robust and efficient gene-engineering tool) in other aquaculture fish species.