Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China.
Department of Animal and Poultry Production, Faculty of Environmental Agricultural Sciences, Arish University, El-Arish, Egypt.
Methods Mol Biol. 2022;2495:295-323. doi: 10.1007/978-1-0716-2301-5_16.
The myostatin (MSTN) gene has shown to play a critical role in the regulation of skeletal muscle mass, and the translational inhibition of this gene has shown increased muscle mass, generating what is known as "double-muscling phenotype." Disruption of the MSTN gene expression using the CRISPR/Cas9 genome-editing system has shown improved muscle development and growth rates in livestock species, including sheep and goats. Here, we describe procedures for the generation of MSTN knockout sheep and goats using the microinjection approach of the CRISPR/Cas9 system, including the selection of targeting sgRNAs, the construction of CRISPR/Cas9 targeting vector, the in vitro examination of system efficiency, the in vivo targeting to generate MSTN knockout founders, the genomic and phenotypic characterization of the generated offspring, and the assessment of off-target effects in gene-edited founders through targeted validation of predicted off-target sites, as well as genome-wide off-target analysis by whole-genome sequencing. Editing the MSTN gene using the CRISPR/Cas9 system might be a rapid and promising alternative to promote meat production in livestock.
肌肉生长抑制素（MSTN）基因在调节骨骼肌质量方面起着关键作用，该基因的翻译抑制已显示出增加肌肉质量，从而产生所谓的“双肌表型”。使用 CRISPR/Cas9 基因组编辑系统破坏 MSTN 基因表达已显示出在绵羊和山羊等家畜中改善了肌肉发育和生长速度。在这里，我们描述了使用 CRISPR/Cas9 系统的微注射方法生成 MSTN 敲除绵羊和山羊的程序，包括靶向 sgRNA 的选择、CRISPR/Cas9 靶向载体的构建、系统效率的体外检测、用于生成 MSTN 敲除种羊的体内靶向、对产生的后代进行基因组和表型特征分析，以及通过对预测的脱靶位点进行靶向验证以及通过全基因组测序进行全基因组脱靶分析来评估基因编辑种羊中的脱靶效应。使用 CRISPR/Cas9 系统编辑 MSTN 基因可能是促进家畜肉类生产的一种快速而有前途的替代方法。
