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 

作者机构：State Key Laboratory of Animal Nutrition and Key Laboratory of Animal GeneticsBreeding and Reproduction of Ministry of Agriculture and Rural Affairs of ChinaInstitute of Animal SciencesChinese Academy of Agricultural SciencesBeijing 100193China Genome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120China Key Laboratory of Agricultural Animal GeneticsBreeding and Reproduction(Huazhong Agricultural University)Ministry of EducationWuhan 430070China 

出 版 物：《Science China(Life Sciences)》 (中国科学（生命科学英文版）)

年 卷 期：2022年第65卷第8期

页      面：1535-1546页

核心收录：

学科分类：0710[理学-生物学] 0830[工学-环境科学与工程（可授工学、理学、农学学位）] 07[理学] 08[工学] 0905[农学-畜牧学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

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) 

主　　题：dual-sg RNA CRISPR-Cas9 ribonucleoproteins transgene-free without monoclonal selection cloned pig 

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.