The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 mRNA and sgRNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals (30%) exhibited large genomic fragment deletions (~5 kb). Furthermore, the body weights of these 3 animals were significantly different (P₀>0.0001, P₁₅=0.001, P₃₀=0.005, P₆₀=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different (P₀>0.0001, P₁₅>0.0001, P₃₀=0.002, P₆₀=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time- and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

CRISPR / Cas9系统已被广泛用于工程基因位点，以在动物模型中产生基因敲除，插入和点突变。然而，在动物中已报道的许多突变是小的插入或缺失。这项研究使用CRISPR / Cas9系统通过绵羊中的三个引导RNA诱导MSTN中的大DNA片段缺失。通过在单细胞阶段将Cas9 mRNA和sgRNA都注入胚胎，成功地实现了绵羊MSTN基因的精确基因编辑。在10只编辑过的动物中，有3只（30％）动物表现出大的基因组片段缺失（〜5 kb）。此外，这3只动物的体重也有显着差异（P ₀ > 0.0001，P ₁₅ = 0.001，在缺失较大的羔羊和野生型羔羊之间，P ₃₀ = 0.005，P ₆₀ = 0.027）。此外，与野生型相比，编辑后的羔羊也有显着差异（P ₀ > 0.0001，P ₁₅ > 0.0001，P ₃₀ = 0.002，P ₆₀ = 0.011）。这些结果表明，所产生的MSTN敲除绵羊是可靠且有效的动物模型，需要进一步研究。此外，该方法省时省力，并且对于创建用于农业，生物学和医学的动物模型是有效的。