Genetically modified pigs have significant value in agricultural and biomedical applications. However, some areas still need to be improved in the process of generating genetically modified pigs:
- It is difficult to quickly obtain a large number of pig individuals with the same genotype.
- Plasmid DNAs can be randomly integrated into the genome of target cells.
- The monoclonal selection process can reduce the quality of donor cells.
Recently, researchers from the Institute of Animal Science and the Institute of Agricultural Genomics of the Chinese Academy of Agricultural Sciences in Shenzhen published a research paper titled “A transgene-free method for rapid and efficient generation of edited pigs accurately without monoclonal selection” in Science Life Sciences in China. They developed an efficient editing system named dual-sgRNA/CRISPR-Cas9 ribonucleoprotein-enriched reporter RNA (RE-DSRNP) for the rapid generation of gene-modified transgene-free cloned pigs, and applied this RE-DSRNP method to establish a porcine model of male reproductive disorder by editing WIP1 uncomfortable.
Researchers used dual sgRNA to improve editing accuracy and efficiency; and used CRISPR-Cas9 ribonucleoprotein (RNP) editing system for transgene-free editing, which successfully avoided the risk of random plasmid DNA integration, and with low off-target effect, low toxicity. They also combined the reporter RNA enrichment system with DSRNP, which eliminated the monoclonal cell selection process based on further improved editing efficiency, thus shortening the cell culture time. of the donor from three to four weeks for one week, and significantly reducing the proportion of donor cells with apoptosis and chromosomal aneuploidy.
In order to verify the application of RE-DSRNP in the establishment of genetically modified cloned pigs, the researchers used the RE-DSRNP system to target the WIP1 in the Meishan pig, a local pig breed in China known for its high reproductive performance. The results showed that: among 32 weaned cloned pigs, 31 (97%) carried WIP1 modifications, and 15 (47%) were homozygous for deletion of the engineered fragment (-38 bp/-38 bp), and no off-target events were detected. the WIP1– the modified pigs showed male reproductive disorders. Breeding boars play a central role in pig farming and commercial production, and have a decisive impact on the fertility of sows. In this article, the WIP1 Genetically modified pig model has been established for the first time, which is of great significance for the in-depth study of male reproductive mechanism.
Somatic cell nuclear transfer is the common technology for generating genetically modified animals for animal breeding and medical model development. The RE-DSRNP system developed in this article will greatly reduce off-target and unexpected effects, greatly improve efficiency and shorten time. It is expected to promote the development of organic breeding and medical models of pigs and other animals, and has broad application prospects. The strong editing performance of RE-DSRNP in a large animal and its marked reduction in the time required to produce SCNT donor cells support its application prospects for rapidly generating transgene-free cloned animal populations.
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