Vet World   Vol.18   December-2025  Article - 10 

Synergistic enhancement of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 -mediated gene editing in porcine zygotes through combined lipofection and electroporation of cationic lipid-packaged ribonucleoproteins

Qingyi Lin¹ , Takeshige Otoi^1,2,3 , Oky Setyo Widodo⁴ , Theerawat Tharasanit⁵ , Kaywalee Chatdarong⁵ , Zhao Namula^1,6 , Maki Hirata^1,6 , Aya Nakai¹ , Yuichiro Nakayama¹ , Megumi Nagahara^1,6 , and Fuminori Tanihara^1,6

Background and Aim: Genetically engineered pigs are invaluable biomedical models for xenotransplantation and the study of human diseases. Although electroporation (EP) and lipofection are individually effective for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) delivery, their combined application in porcine embryos has not been systematically evaluated. This study aimed to determine whether packaging Cas9-guided RNA complexes in cationic lipids enhances EP-mediated gene editing efficiency without compromising embryonic development. 

Materials and Methods: Porcine zygotes with their zona pellucida removed were edited using RNPs targeting beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) or growth hormone receptor (GHR) genes. Four treatment groups were tested: (1) EP with RNPs (EP), (2) EP with lipofectamine-packaged RNPs (EPL), (3) transfection with lipofectamine-packaged RNPs before EP (TL + EPL), and (4) EP followed by lipofection (EPL + TL). Blastocyst formation was evaluated morphologically, and mutation rates were assessed by Sanger sequencing followed by tracking of indels by decomposition (TIDE) analysis. 

Results: Blastocyst formation rates were comparable across all treatments, indicating that lipofectamine packaging and EP caused no detectable cytotoxicity. For B4GALNT2, no mutations were induced by EP alone, whereas TL + EPL treatment significantly increased total and mosaic mutation rates (p < 0.05). For GHR, the total mutation and mosaic mutation rates were likewise higher in TL + EPL compared with EP, although mutation efficiency (indel percentage per edited embryo) remained unchanged. These results suggest that pre-EP lipofection promotes RNP uptake by facilitating lipid-membrane interactions that are potentiated by subsequent membrane destabilization through EP. 

Conclusion: Packaging RNPs in cationic lipids and applying sequential lipofection followed by EP significantly enhances CRISPR/Cas9-mediated mutagenesis in porcine zygotes without affecting developmental competence. This dual-delivery approach provides a simple, reproducible, and low-toxicity workflow for generating gene-edited embryos, with potential applicability to large-animal biomedical models. 

Keywords: cationic lipid, CRISPR/Cas9, electroporation, genome-editing efficiency, lipofectamine, porcine zygote, xenotransplantation.

How to cite this article: Lin Q, Otoi T, Widodo OS, Tharasanit T, Chatdarong K, Namula Z, Hirata M, Nakai A, Nakayama Y, Nagahara M, and Tanihara F (2025) Synergistic enhancement of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene editing in porcine zygotes through combined lipofection and electroporation of cationic lipid-packaged ribonucleoproteins, Veterinary World, 18(12): 3806–3814.

Received: 21-09-2025   Accepted: 07-11-2025   Published online: 10-12-2025

Corresponding author: Takeshige Otoi    E-mail: otoi@tokushima-u.ac.jp

DOI: 10.14202/vetworld.2025.3806-3814

Copyright: Lin, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.