Su Xiaoping, Cui Kuiqing, Du Shanshan, Li Hongli, Lu Fenghua, Shi Deshun, Liu Qingyou
Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530004, China.
In Vitro Cell Dev Biol Anim. 2018 May;54(5):375-383. doi: 10.1007/s11626-018-0236-8. Epub 2018 Mar 19.
Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.
肌肉生长抑制素（MSTN）是一种由生长分化因子8（GDF8）编码的蛋白质，主要在骨骼肌中表达，并对肌肉的发育和再生起负调控作用。因此，缺乏肌肉生长抑制素的动物表现出双肌 phenotype。CRISPR/Cas9系统已被证明是一种有效的基因组编辑工具，并已应用于许多模式生物（如黑腹果蝇、斑马鱼、小鼠、大鼠、绵羊和人类）细胞的基因修饰。在这里，我们使用CRISPR/Cas9系统对德保猪和沼泽水牛的成纤维细胞和胚胎中的GDF8基因进行了编辑。CRISPR/Cas9介导的猪成纤维细胞突变效率高达87.5%，水牛成纤维细胞突变效率为78.9%。然后，我们通过在猪和水牛的成纤维细胞中用G418筛选，获得了在GDF8基因特定位点发生突变的单细胞克隆。此外，Cas9/gRNA介导的突变频率在猪的胞浆内精子注射胚胎和水牛的体外受精胚胎中分别为36%和25%。我们的工作表明，Cas9/gRNA系统是一种用于德保猪和沼泽水牛培养细胞和胚胎基因组编辑的高效快速工具。这些结果有助于建立一种能产生更多肉的新动物品系。
