The global demand for fish and fisheries products has increased over the last few decades because of their high protein and highly unsaturated fatty acid contents. To cope with this increase, it has become necessary to use limited resources more efficiently. The myostatin (mstn) gene, a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth by inhibiting the proliferation and differentiation of muscle fibers. In mstn-null fish, muscle growth is accelerated, leading to increased muscle mass, a phenomenon known as the double-muscling phenotype. The Black Sea trout (Salmo labrax) is an economically important fish species in Türkiye. Therefore, to produce better-growing S. labrax, we knocked out the mstn gene using the CRISPR/Cas9 genome editing technique.
The mstn gene in Salmo labrax was cloned and sequenced. Two single guide RNAs (sgRNA) targeting the first exon of the mstn were designed using the CRISPOR database. Oligonucleotides were cloned into a pT7-gRNA vector. In vitro transcription was performed using the Megascript T7 Kit. Embryos were injected with a mixture of 400 pg Cas9 protein and 200 pg sgRNA. Subsequently, they were incubated in 10 oC water. Genomic DNA was extracted from caudal veins of mutant (mstn-) and wild type (mstn+) fish. The target region was amplified by PCR. The products from mstn- and mstn+ fish were cloned into the pCRTM4-TOPO® vector using the TOPO TA Cloning Kit and were sequenced. T7 endonuclease assay was performed and digested products were separated by agarose gel electrophoresis.
Hatching rate and larval growth rate of mstn- Salmo labrax were lower than those of mstn- Salmo labrax. Mutation rate was 35.4±2.08% (Table). Six-months old mstn- fish was approximately %60 heavier than wild type fish (Figure).
We successfully knocked out mstn gene in Salmo labrax at the first time and this would be helpful to produce faster-growing fish in the future.