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

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 e ciently 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 modi cation 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 modi ed CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modi ed 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 broblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs broblast cells carrying the MSTN premature termination and veri ed 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.

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

“…Gene sequencing showed that 33.3% (4/12) of single-cell colonies had biallelic ABE-mediated Ato-G conversions at the target locus. This e ciency is comparable to that of CRISPR/Cas9 in generating geneknockout pig cells conducted in our previous studies, suggesting that the ABE system is a practical and feasible approach to generating genetically modi ed porcine cell colonies (Zhu et al 2018;Wei et al 2020;Pan et al 2021). More importantly, RT-PCR and Western blotting tests showed that ABE-mediated single-base mutations in the conservative splice acceptor in the intron 5 of GHR successfully induced exon 6 skipping at the RNA level and gene knockout at the protein level.…”

“…PCR amplicons were puri ed using universal DNA puri cation kit (Tiangen) and sent to the GENEWIZ company for Sanger and next generation sequencing according to our previous study (Pan et al 2021). For next generation sequencing, amplicons were ligated to adapters and sequencing was performed on an Illumina MiSeq 2x300bp sequencing platform.…”

“…Design and construction of the ABE plasmid were performed according to our previous studies (Zhu et al 2018;Wei et al 2020;Pan et al 2021). The sgRNAs used in this study were designed as described below, and were produced by BGI company.…”

“…Procedures used for the isolation, cultivation, and transfection of kidney broblasts from newborn Bama minipigs were based upon our previous studies (Zhu et al 2018;Wei et al 2020;Pan et al 2021). After deep anesthetization, the newborn piglet was euthanized and both kidneys were removed and minced in Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA).…”

“…In human cells, previous studies have shown that the editing window spanning of modi ed ABE vector (PX-ABEmaxAW) was located at positions 4 to 8 (counting the protospaceradjacent motif (PAM) as positions 21 to 23) (Pan et al 2021). To assess the base-editing activity of the modi ed ABE editor on Bama minipig cells, we selected several genome-editing sites, including sites of genes related to economic trait locus (myostatin gene, MSTN) and those of genes related to human disease, gene for coding Iroquois homeobox 3 (IRX3) and the gene for coding dystrophin (DMD), which are being used for gene editing using CRISPR/Cas9 system in our team.…”