A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection

文献类型： 外文期刊

第一作者： Xu, Kui

作者： Xu, Kui;Zhang, Xiuling;Liu, Zhiguo;Ruan, Jinxue;Xu, Changjiang;Che, Jingjing;Fan, Ziyao;Mu, Yulian;Li, Kui;Xu, Kui;Zhang, Xiuling;Liu, Zhiguo;Ruan, Jinxue;Xu, Changjiang;Che, Jingjing;Fan, Ziyao;Mu, Yulian;Li, Kui;Xu, Kui;Li, Kui;Ruan, Jinxue

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关键词： dual-sgRNA; CRISPR-Cas9 ribonucleoproteins; transgene-free; without monoclonal selection; cloned pig

期刊名称：SCIENCE CHINA-LIFE SCIENCES （ 影响因子：10.372； 五年影响因子：7.587 ）

ISSN： 1674-7305

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收录情况： SCI

摘要： Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3-4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP's strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.

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