RE-DSRNP mediated efficient editing system and its application in generation of WIP1 gene-edited male reproductive disorder pig model (IMAGE)

Science China Press

RE-DSRNP mediated efficient editing system and its application in generation of WIP1 gene-edited male reproductive disorder pig model

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The reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins (RE-DSRNP) method uses dual-sgRNA in combination with reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), which incorporates the precise targeting of dual-sgRNA systems with the increased editing rates but reduced off-target effects and cytotoxicity associated with reporter RNA-enriched RNPs, without the need for introducing transgenes, thus eliminating the need for the selection of monoclonal cells and thereby reducing the generation time of donor cells from 3-4 weeks to 1 week. RE-DSRNP was used to produce cloned pigs bearing a deletion edit of the 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.

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