Key Takeaways & Executive Findings
- •• • Three monoclonal IgA-knockout porcine fetal fibroblast lines were generated with precise deletions of 1044 bp (heterozygous), 1043 bp (homozygous), and 1039 bp (homozygous) in the CH1-CH3 region, demonstrating high editing efficiency and genotype diversity without transgene integration. • • SCNT using pooled donor cells yielded 400 fused cells from 500 enucleated oocytes (80% fusion rate), and transfer of 300 embryos into a surrogate sow resulted in a 28-day pregnancy confirmation and birth of six live F0 piglets after 143 days of gestation, achieving a 2% birth rate per transferred embryo. • • Genotyping of F0 piglets confirmed two heterozygotes (4010#, 4015#) and four homozygous knockouts, with exactly two piglets per genotype (1044, 1043, 1039 bp deletions), validating the deliberate mixing of donor cells to obtain multiple genotypes in a single pregnancy. • • The IRE-DSRNP method bypasses plasmid DNA, eliminating resistance gene integration and reducing off-target cleavage risks, while shortening the cell line acquisition period from 3–4 weeks to a rapid, transgene-free workflow, directly addressing biosafety and timeline bottlenecks in transgenic pig production.
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Abstract
The generation of genetically edited pigs via somatic cell nuclear transfer (SCNT) has historically relied on plasmid-based CRISPR/Cas9 systems, which introduce resistance genes, risk off-target effects from prolonged editing, and require 3–4 weeks of in vitro selection. This study presents a transgene-free, rapid strategy using the IRE-DSRNP method to edit monoclonal porcine fetal fibroblasts. Three IgA-knockout cell lines were obtained with large deletions in the CH1-CH3 region: 1044 bp (heterozygous), 1043 bp (homozygous), and 1039 bp (homozygous). These cells were pooled and used as donor nuclei for SCNT. From 880 fresh oocytes, 660 mature oocytes were selected, 500 underwent enucleation and nuclear transfer, yielding 400 fused cells; 300 embryos were transplanted into a surrogate sow. Pregnancy was confirmed at 28 days, and after 143 days of gestation, six F0 piglets were born. Genotyping revealed two heterozygotes (4010#, 4015#) and four homozygous knockouts, with two piglets per genotype (1044, 1043, 1039 bp deletions). This approach eliminates plasmid integration, reduces off-target risks, and shortens the timeline for producing IgA-deficient Bama pig models, offering a robust platform for disease modeling and xenotransplantation research.
1. Introduction
Plasmid-based CRISPR/Cas9 editing has been the predominant method for generating genetically edited porcine embryonic fibroblasts for SCNT. However, this approach introduces antibiotic resistance genes, risks persistent DNA integration that elevates off-target effects and random mutations, and demands 3–4 weeks of in vitro selection, increasing apoptosis and chromosomal aberration rates. These limitations compromise biosafety and efficiency, impeding the reliable production of gene-edited pigs for biomedical and agricultural applications.
The IRE-DSRNP method delivers Cas9 protein and sgRNA directly as ribonucleoproteins, eliminating plasmid DNA and reducing cytotoxicity and off-target activity. By applying this transgene-free strategy to monoclonal porcine fetal fibroblasts, we achieved precise large-fragment deletions in the IgA CH1-CH3 region. The edited cells were then used as donor nuclei for SCNT, enabling the rapid generation of IgA-knockout Bama pigs with multiple genotypes in a single surrogate pregnancy, thereby overcoming the biosafety and timeline bottlenecks of conventional plasmid editing.
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Kun Liu, Nan Huang, Chuanxiang Ding, Qiaoli Lang, Hongyu Chen, Hao Liang, Rendong Fang, Liangpeng Ge, Xi Yang (2025). A simple, rapid, and transgene-free strategy for the generation of transgenic pigs via precise editing of monoclonal porcine fetal fibroblasts. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2025044
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Frequently Asked Questions
What is the fusion efficiency and embryo development rate of the SCNT procedure using IRE-DSRNP-edited fibroblasts?
From 880 fresh oocytes, 660 mature oocytes were selected, and 500 underwent enucleation and nuclear transfer, yielding 400 fused cells (80% fusion rate). After activation, 300 embryos were transplanted, resulting in a 28-day pregnancy and six live births after 143 days, corresponding to a 2% birth rate per transferred embryo.
How does the IRE-DSRNP method reduce off-target effects compared to plasmid-based editing?
The IRE-DSRNP method delivers Cas9 protein and sgRNA as a ribonucleoprotein complex, avoiding plasmid DNA integration and persistent expression. This minimizes continuous editing, off-target cleavage, and random mutations, as confirmed by off-target analysis in the pig genome, whereas plasmid systems risk prolonged editing and resistance gene insertion.
What genotypes were obtained in the F0 generation, and how were multiple genotypes achieved in a single pregnancy?
Three distinct genotypes were obtained: 1044 bp deletion (heterozygous), 1043 bp deletion (homozygous), and 1039 bp deletion (homozygous). By intentionally mixing the three monoclonal cell lines as donor cells for SCNT, six F0 piglets were born, comprising two heterozygotes (4010#, 4015#) and four homozygous knockouts, with two piglets per genotype.
What is the timeline advantage of the IRE-DSRNP method over conventional plasmid editing for generating transgenic pigs?
Plasmid editing typically requires 3–4 weeks of in vitro selection to obtain positive cell lines, with increased risks of apoptosis and chromosomal aberrations. The IRE-DSRNP method bypasses plasmid transfection and drug selection, enabling rapid generation of edited monoclonal fibroblasts and shortening the overall timeline to produce IgA-knockout pigs.
What are the biosafety and clinical implications of eliminating resistance genes in donor cells for SCNT?
Eliminating resistance genes removes the risk of horizontal gene transfer and drug resistance in recipient animals, addressing biosafety concerns. This is critical for producing transgenic pigs intended for xenotransplantation or human disease models, where transgene-free editing reduces immunogenicity and regulatory hurdles.
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