Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, 528225, Foshan, China.
Guangdong Provincial Engineering and Technology Research Center for Gene Editing, School of Medicine, Foshan University, 528225, Foshan, China.
Biotechnol Lett. 2021 Sep;43(9):1699-1714. doi: 10.1007/s10529-021-03159-1. Epub 2021 Jun 30.
Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.
巴马小型猪是中国培育的一种独特的小型猪。它们的优良特性包括肉质鲜美、适应性强、能耐受粗饲料、应激能力高。不良特性是瘦肉率低、脂肪含量高、生长速度慢、饲料转化率低。利用 CRISPR/Cas9 基因组编辑技术有效地敲除了对肌肉生成有负调控作用的肌肉生长抑制素基因（MSTN），有效促进了猪肌肉生长，提高了猪的瘦肉率。然而，CRISPR/Cas9 基因组编辑技术是基于 DNA 双链断裂实现的随机突变，可能引发基因组脱靶效应和染色体重排。CRISPR/Cas9 在提高猪经济性状中的应用引起了生物安全方面的担忧。基于 CRISPR/Cas9 开发的碱基编辑器（BE），如胞嘧啶碱基编辑器（CBE），可以有效地在不依赖于 DNA 双链断裂的情况下实现单个碱基的靶向修饰。因此，该方法在猪的遗传改良中具有更大的安全性。本研究旨在利用改良的 CBE 生成巴马小型猪 MSTN 敲除细胞。研究结果表明，构建的“all-in-one”-改良 CBE 质粒实现了巴马小型猪成纤维细胞 MSTN 靶标中单个 C·G 碱基对定向转换为 T·A 碱基对。成功构建了多个携带 MSTN 提前终止的巴马小型猪成纤维细胞单克隆细胞，并验证了没有检测到基因组脱靶效应。本研究为进一步应用体细胞克隆技术构建仅携带单个碱基突变的 MSTN 编辑巴马小型猪提供了基础，在很大程度上避免了生物安全风险，为巴马小型猪其他遗传位点的碱基编辑提供了经验和参考。
