Abstract
The myostatin (MSTN) gene is important because of its role in regulation of skeletal muscle growth in all vertebrates. In this study, CRISPR/Cas9 was utilized to successfully target the channel catfish, Ictalurus punctatus, muscle suppressor gene MSTN. CRISPR/Cas9 induced high rates (88–100%) of mutagenesis in the target protein-encoding sites of MSTN. MSTN-edited fry had more muscle cells (p < 0.001) than controls, and the mean body weight of gene-edited fry increased by 29.7%. The nucleic acid alignment of the mutated sequences against the wild-type sequence revealed multiple insertions and deletions. These results demonstrate that CRISPR/Cas9 is a highly efficient tool for editing the channel catfish genome, and opens ways for facilitating channel catfish genetic enhancement and functional genomics. This approach may produce growth-enhanced channel catfish and increase productivity.
Highlights
Fish comprise the most diverse group among the vertebrates, a much smaller number of fish species is used for research and commercial purposes
Based on the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) from Streptococcus pyogenes[42, 43], the co-delivery of endonuclease Cas[9] combined with a synthetic small guide RNA targeting certain gene(s) into eukaryotic cells can edit the genome by stimulating a double-strand break (DSB) at a desired site(s)
Unlike zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), small guide RNA (sgRNA) is the only component that needs designation for each genomic target, thereby significantly simplifying the design and lowering the cost of gene editing compared to the protein-based target recognition platforms
Summary
Fish comprise the most diverse group among the vertebrates, a much smaller number of fish species is used for research and commercial purposes. A new gene editing system has been applied with higher targeting efficiency and lower cell toxicity, known as clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas[9]). CRISPR mutagenesis was performed to edit various genes (including MSTN) in some aquaculture species[55,56,57,58,59]. We used CRISPR zygote microinjection to knockout MSTN gene in channel catfish, and determined the effects of knockout on growth. We aim to produce growth-enhanced lines of channel catfish with CRISPR technology These gene-edited lines would be fully characterized for growth rate, feed efficiency and disease resistance and compared to genotypes currently used in aquaculture
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