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. The pigs also have the potential to be used as medical disease models (Zhu et al., 2018). However, 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).
Traditional genetic selection in pigs involves long breeding cycles, high costs, and limited genetic resources, particularly regarding increased yield, improvements in quality, or enhancement of stress tolerance. In contrast, genome-editing technology represented by CRISPR/Cas9 effectively modifies the genome of pigs using modified cells carrying the target genes as nuclear donors for somatic cell cloning; this method can achieve stable improvement of one or more production traits (Petersen et al., 2017; Ruan et al., 2017). Hence, the technology is expected to have significant potential for genetic improvement of pigs. By generating a double-strand break at the target genetic locus, CRISPR/Cas9 genome editing technology relies on the DNA repair mechanism of the cells to introduce a certain number of base insertions or deletions at the break sites, efficiently disrupting gene expression (Zhao et al., 2019). However, the editing results triggered by CRISPR/Cas9 are random and rely on DNA double-strand breaks; this may cause genomic off-target effects, deletion of large gene fragments, abnormal chromosomal structure, and even whole chromosome deletions, leading to unpredictable results in the edited cells (Kosicki et al., 2018; Zuccaro et al., 2020). Because of these shortcomings, the application of CRISPR/Cas9 to improve economic traits in pigs has led to biosafety concerns.
To ensure a high precision of genome editing, base editor (BE) technology that achieves single-base targeted mutations has emerged. In 2016, Liu and colleagues fused the Cas9n protein D10A (which only leads to DNA single-strange cleavage) with rat cytosine deaminase, rAPOBEC (which catalyzes the deamination of C into U, and U is recognized as T during DNA replication), and uracil DNA glycosylase inhibitor, UGI (which prevents uracil glycosylase from glycosylation of U to cause base excision repair) (Komor et al., 2016). This fusion protein specifically targets C·G base pairs for mutation to T·A base pairs under the guidance of single-stranded guide RNA (sgRNA); the protein is also known as cytosine base editor (CBE). CBE does not produce DNA double-strand breaks, rather only resulting in the targeted mutation of a single C·G base pair to a T·A base pair, and thus is more precise than the original CRISPR/Cas9 genome editing technology (Komor et al., 2016). CBE is expected to be safer in genetic improvement of pigs. To date, the application of CBE in genetic modification of pigs has achieved several breakthroughs. Although BEs have been successfully used to produce genetically engineered pigs (Li et al., 2018; Xie et al., 2019; Wang et al., 2020), the BEs used in the past, such as BE3, were shown to cause a high proportion of DNA and RNA off-target effects (Komor et al., 2016). Improved versions of BE (e.g., YE1-BE4maxNG) with the systematic transformation through protein engineering technology have been constructed; these have a lower level of RNA editing activity and fewer insertion/deletion mutations while maintaining the efficiency of base editing (Doman et al., 2020). These improved versions of BEs will become increasingly important in the genetic improvement of pigs.
The most significant unfavorable characteristics of Bama minipigs are the low lean meat percentage and high fat content. Our research team particularly aims to utilize genome editing technology to increase the lean-meat yield of Bama minipigs by essentially eliminating the key genes that inhibit the muscle growth of the animals. Myostatin gene (MSTN), also known as growth differentiation factor 8 (GDF8), is mainly expressed in skeletal muscle and has a negative regulatory effect on muscle development and regeneration. The well-known double-muscled hips in Belgian Blue Cattle is a significant developmental phenotype produced by a loss-of-function mutation in MSTN (Grobet et al., 1997). Studies have shown that MSTN-knockout mediated by zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 replicate the phenotype of double-muscled hips in rabbits, cows, goats, sheep, and pigs, thereby significantly increasing the lean meat percentage and reducing the fat content of the animals (He et al., 2018; Lv et al. 2016; Wang et al., 2016). Thus, MSTN is an ideal target for preparing Bama minipigs with significantly increased lean meat percentage.
In view of the fact that base editing has more precise genome editing effects and greater safety in the genetic improvement of livestock, this study aimed to use modified CBE to prepare MSTN-knockout cells of Bama minipigs by firstly constructing an “all-in-one” modified CBE plasmid and verifying that this plasmid achieved the directional conversion of a single C·G base in the editing window to T·A base pairs at multiple gene loci in the cells of Bama minipigs. Subsequently, four potential targets (CBE introduces premature stop codons to disrupt MSTN function) were designed according to the characteristics of the editing window of CBE, followed by comparing their activities to select the most efficient target among them for the preparation of single-cell colonies. Lastly, the prepared single-cell colonies and base-editing effects were identified, and the genomic off-target effects on the single-cell colonies with expected base edits were analyzed. We constructed and used the modified CBE plasmid to successfully prepare single-cell colonies of Bama minipigs carrying the MSTN premature termination and without genomic off-target effects. 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 a reference for the base editing of other genetic loci in Bama minipigs.