Gene-edited pigs have important agricultural and biomedical applications. However, there are still some troubles in the production process of gene-edited pigs: (1) it is difficult to quickly obtain a large number of pigs with the same genotype; (2) plasmid DNA may be randomly integrated into the genome of the target cell; and (3) the monoclonal selection process may reduce the quality of donor cells.
Recently, researchers at the Shenzhen Institute of Animal Science and the Institute of Agricultural Genomics of the Chinese Academy of Agricultural Sciences published a research paper titled "A Non-GMO Method that Can Quickly and Efficiently Breed Gene-Edited Pigs Without Single Clonal Selection" in the journal Sciences of China: Life Sciences. They developed an efficient RNA editing technique called The Report RNA-enriched Bi-Guide RNA Nuclear Protein (RE-DSRNP) for rapid construction of cloned pigs without foreign DNA gene editing, and applied this method to establish a model of male reproductive disorder in pigs by editing the WIP1 gene.
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In order to improve the accuracy and efficiency of editing, this paper uses dual-guided RNA technology to use CRISPR-Cas9 ribonucleoprotein (RNP) editing system for gene-free editing, which successfully avoids the risk of random integration of plasmid DNA into the genome, and has low off-target effect and lower toxicity. This study will also report that the RNA enrichment system binds to the bibo-guided RNA nuclear protein (DSRNP), eliminating the monoclonal cell selection process while further improving the editing efficiency, thereby shortening the donor cell culture time from 3-4 weeks to 1 week, significantly reducing the proportion of donor cells undergoing apoptosis and aneuploidy chromosomes.
In order to verify the application effect of RE-DSRNP in establishing gene-edited cloned pigs, the researchers used the Meishan pig as a material to use the RE-DSRNP system to target the editing of its WIP1 gene. The results showed that of the 32 weaned gene-edited pigs obtained, 31 (97%) carried the WIP1 gene, 15 (47%) were pure zygotes with dna fragment deletion (-38 bp/-38 bp) of the design, all cloned pigs were not detected off-target, and the WIP1-edited pigs showed male reproductive disorders. Breeding boars play a pivotal role in pig breeding and commercial production, and have a decisive influence on the fertility of sows. This paper constructs the BREEDING MODEL OF WIP1 gene-edited pigs for the first time, which is of great significance for the in-depth study of male reproductive mechanism.
RE-DSRNP-mediated efficient editing system and its application in generating WIP1 gene-edited pig models| references[2]
Somatic cloning gene editing technology is currently the mainstream technology for breeding gene-edited animals, and is commonly used in animal breeding and the development of medical models. The RE-DSRNP system developed in this paper can significantly reduce the impact of off-target and unintended effects, greatly improve efficiency and shorten time. It is expected to promote the establishment and development of biological breeding and medical models of pigs and other animals, and has broad application prospects. In large animals, the robust gene-editing performance of RE-DSRNP and its significant reduction in the time required to generate SCNT donor cells support its application prospects in rapidly generating non-GMO cloned animal populations.
Compilation: Four seven
Edit: Crispy fish
Typography: Yin Ningliu
Image source: "Weird Town"
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[1] https://www.eurekalert.org/news-releases/947576
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