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Application of the modified cytosine base-editing in the cultured cells of bama minipig. | LitMetric

Application of the modified cytosine base-editing in the cultured cells of bama minipig.

Biotechnol Lett

Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, 528225, Foshan, China.

Published: September 2021

AI Article Synopsis

  • Bama minipigs are a specialized breed known for their adaptability and meat quality, but they have drawbacks like low lean meat and slow growth rates; genetic editing aims to improve these traits.
  • CRISPR/Cas9 technology can increase muscle growth in pigs by targeting the myostatin gene, but it poses risks of unintended genetic changes, raising biosafety concerns.
  • The study successfully used a modified base editor to create specific genetic modifications in Bama minipig cells without causing off-target effects, setting the stage for safer genetic improvements in this breed.

Article 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.

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Source
http://dx.doi.org/10.1007/s10529-021-03159-1DOI Listing

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