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Research Article | 13 Jun 2026

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

Koki Takebayashi1 ORCID , Takeshige Otoi1,2,3,4 ORCID , Manita Wittayarat5 ORCID , Oky Setyo Widodo2 ORCID , Theerawat Tharasanit3 ORCID , Kaywalee Chatdarong3 ORCID , Yuichiro Nakayama1 ORCID , Zhao Namula1,4 ORCID , Megumi Nagahara1 ORCID , Maki Hirata1 ORCID , and Fuminori Tanihara1 ORCID Show more
VETERINARY WORLD | Article No. 11 | pg no. 2406-2417 | Vol. 19, Issue 6 | DOI: 10.14202/vetworld.2026.2406-2417
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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 in vitro fertilization (IVF) were evaluated by collecting zygotes at 8–24 h post-fertilization, followed by centrifugation, fixation, and Hoechst 33342 staining. In the second experiment, the effects of Hoechst 33342 concentration (5 and 10 μg/mL) and exposure duration (10 and 30 min) on embryo development were assessed using live zygotes collected 20 h after fertilization. In the third experiment, zygotes with visible 2PN selected after centrifugation and Hoechst staining were electroporated with guide RNA targeting the TP53 gene. Developmental competence, mutation efficiency, and mosaicism were compared with non-selected control zygotes. 
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. 
                      
Keywords: blastocyst development, CRISPR/Cas9, electroporation, genome editing, Hoechst 33342, mosaicism, porcine embryos, two pronuclei.