Abstract
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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Acknowledgements
This work was 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), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS05).
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Xu, K., Zhang, X., Liu, Z. et al. A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection. Sci. China Life Sci. 65, 1535–1546 (2022). https://doi.org/10.1007/s11427-021-2058-2
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DOI: https://doi.org/10.1007/s11427-021-2058-2