ArticleVeterinary world2026
Selection of two pronuclei zygotes before electroporation does not improve gene editing efficiency or reduce mosaicism in porcine embryos despite optimized timing and Hoechst staining.
Article in Veterinary world, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Background and Aim: Electroporation-mediated delivery of Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 components is an effective approach for genome editing in porcine embryos; however, mosaicism remains a major limitation. Selecting normally fertilized zygotes with two pronuclei (2PN) before electroporation may improve editing uniformity and reduce mosaicism. Therefore, this study investigated the effects of 2PN selection, optimized timing of pronuclear observation, and Hoechst 33342 staining conditions on embryo development and genome editing efficiency in porcine embryos. Materials and Methods: In the first experiment, pronuclear dynamics after Results: The proportion of zygotes with 2PN gradually increased after fertilization and reached the highest level (47.4%) at 20 h post-IVF before significantly declining at 24 h (p < 0.05). Among the staining conditions tested, treatment with 5 μg/mL Hoechst 33342 for 30 min resulted in the highest blastocyst formation rate (14.3%, p < 0.05). Following electroporation, blastocyst development rates did not differ significantly between the 2PN-selected group (6.5%) and the control group (10.8%). Similarly, the biallelic mutation rate and total mutation efficiency were comparable between selected zygotes (78.6% and 85.5%, respectively) and controls (81.8% and 92.5%, respectively). Conclusion: Selection of porcine zygotes with 2PN before electroporation did not improve genome editing efficiency or reduce mosaicism despite optimization of pronuclear observation timing and Hoechst staining conditions. The persistence of mosaicism may be associated with asynchronous DNA replication between maternal and paternal pronuclei. Although 2PN selection was safe for embryo development, additional strategies targeting pronuclear synchronization and replication timing are required to achieve more uniform genome editing in porcine embryos.
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