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

作　　者：Kui Xu Xiuling Zhang Zhiguo Liu Jinxue Ruan Changjiang Xu Jingjing Che Ziyao Fan Yulian Mu Kui Li 

机构地区：[1]State Key Laboratory of Animal Nutrition and Key Laboratory of Animal Genetics,Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China [2]Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,China [3]Key Laboratory of Agricultural Animal Genetics,Breeding and Reproduction(Huazhong Agricultural University),Ministry of Education,Wuhan 430070,China

出　　处：《Science China(Life Sciences)》2022年第8期1535-1546,共12页中国科学（生命科学英文版）

基　　金：supported by the National Natural Science Foundation of China (32072690);the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202006);the Science, Technology and Innovation Commission of Shenzhen Municipality (JCKYZDKY202009);the National Transgenic Breeding Project (2016ZX08010-004);the National Transgenic Breeding Project (2016ZX08006-001);the Agricultural Science and Technology Innovation Program (ASTIP-IAS05)。

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-sg RNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sg RNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs(CRISPR-Cas9ribonucleoproteins), 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. REDSRNP'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.

关 键 词：dual-sg RNA CRISPR-Cas9 ribonucleoproteins transgene-free without monoclonal selection cloned pig 

分 类 号：Q78[生物学—分子生物学] S828[农业科学—畜牧学]