Abstract
Myostatin gene (MSTN) can inhibit the proliferation of myoblast, which in turn promotes muscle growth and inhibits adipocyte differentiation in livestock. MSTN mutation may lead to muscle hypertrophy or double-muscled (DM) phenotype. MSTN mutation animal, such as sheep, dog, and rabbit have been generated through CRISPR/Cas9 technology. However, goats with promising MSTN mutation have not been generated. We designed two sgRNAs loci targetting exon3 of MSTN gene to destroy the MSTN cysteines knots. We got seven goats from seven recipients, in which six were MSTN knocked-out (KO) goats, with a mutation rate of 85.7%. Destroyed cysteine knots caused MSTN structure inactivation. The average body weight gain (BWG) per day of MSTN KO goats was significantly higher than that of wild-type (WT) goats. MSTN KO goats showed abnormal sugar, fat, and protein metabolism compared with wild-type controls (MSTN+/+). Inheritance of mutations was observed in offspring of MSTN KO goats by PCR analysis.
Highlights
Myostatin gene (MSTN) as a member of transforming growth factor β (TGF-β) family is called the growth differentiation factor 8 (GDF-8) [1]
MSTN protein is expressed in almost all types of tissues, and the expression level is relatively high in skeletal muscle, which facilitates its role as a negative regulatory factor in the proliferation of myoblast [2]
PCR results showed that no mutation was detected in these putative off-target sites (POTS), indicating that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNA (sgRNA) system did not induce undesirable off-target effect in MSTN KO goats
Summary
Myostatin gene (MSTN) as a member of transforming growth factor β (TGF-β) family is called the growth differentiation factor 8 (GDF-8) [1]. With the advantages of simple operation, low cost, low off-target rate and high efficiency, CRISPR/Cas has been widely used for different purposes, such as disease model construction [5], genetic screens [6], gene function characterization [7], and so on. CRISPR/Cas has been used to generate MSTN mutation animals, such as rabbit [8] and sheep [9]. Construction of FGF5 and MSTN double knockout goat mutant by CRISPR/Cas technology has been reported by a previous study [10]. Exon 3 of MSTN gene, which has not been targetted before, was selected as the target site to generate MSTN mutation goats using CRISPR/Cas technology. The mutation rate, muscle mass yield, genetic stability, and animal health status were discussed
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