Abstract
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.
Highlights
Bama minipigs are a unique breed originating from Bama Yao Autonomous County in Guangxi, China
This study provides a foundation for further application of somatic cell cloning to construct myostatin gene (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
To test the activity of the constructed “all-in-one” modified cytosine base editor (CBE) plasmid on the cells of Bama minipigs, four endogenous genetic loci were selected for activity testing, the target genes being apolipoprotein A5 (APOA5) and the low-density lipoprotein receptor (LDLR) for the construction of human disease models and the CD163 and MSTN genes for the improvement of economic traits in pigs
Summary
Bama minipigs are a unique breed originating from Bama Yao Autonomous County in Guangxi, China. The breed has favorable characteristics including delicious meat, strong adaptability, tolerance to rough feed, and a high level of stress tolerance. Bama minipigs have played an important role in local economic growth and agriculture, and thereby in poverty reduction. In addition to food consumption, because of its small size, Bama minipigs have become a well-known local specialty throughout the country, with a high value in the area’s economic development (Zhu et al, 2020a; b). Characteristic traits of the breed include early sexual maturity, large litters, and a high degree of inbreeding. Similar to other indigenous pig breeds in China, Bama minipigs have unfavorable characteristics such as low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio, hindering the breed’s application and promotion (Zhu et al, 2020a; b)
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