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

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.