Abstract
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
Highlights
Traditional pig breeding is limited by the long breeding cycle and insufficient genetic resources, highlighting the potential value of genetic modification that can significantly improve a specific heritable production trait in pigs in one generation
An Adenine base editor (ABE) system is composed of an editor for base editing and an single guide RNA (sgRNA) for target recognition; these are fused into a plasmid to improve cell transfection and editing efficiency
The ABE sequence ABEmaxAW modified for reducing off-target effects and improving editing activity was fused with the sgRNA expression cassette in the vector (Fig. 1A)
Summary
Traditional pig breeding is limited by the long breeding cycle and insufficient genetic resources, highlighting the potential value of genetic modification that can significantly improve a specific heritable production trait in pigs in one generation. Researchers have created a variety of genetically modified pigs with excellent production traits, resulting in significant improvements in feed utilization, lean meat percentage, disease resistance, and healthy fatty acid composition In addition to the potential applications in agriculture, genetically modified pigs have significant biomedical uses as ideal animal models (Petersen 2017; Perleberg et al 2018; Yan et al 2018; Zhu et al 2018; Zhao et al 2019; Koppes et al 2020). Pigs are highly fecund, with early sexual maturation (5–8 months), short generation interval (10–12 months), large litters
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