Efficient gene editing of pig embryos by combining electroporation and lipofection

Vet World Vol.17 November-2024 Article - 30

Research Article

Veterinary World, 17(11): 2701-2707

https://doi.org/10.14202/vetworld.2024.2701-2707

Qingyi Lin^1,2, Nanaka Torigoe^1,2, Bin Liu^1,2, Yuichiro Nakayama^1,2, Aya Nakai^1,2, Zhao Namula^1,3, Megumi Nagahara^1,2, Fuminori Tanihara^1,2, Maki Hirata^1,2, and Takeshige Otoi^1,2

1. Bio-Innovation Research Center, Tokushima University, Tokushima, Japan.
2. Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.
3. Department of Animal Reproduction, College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, China.

Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences.

Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls.

Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates.

Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing.

Keywords: electroporation, guide RNA sequence, lipofection, pig embryo.

How to cite this article: Lin Q, Torigoe N, Liu B, Nakayama Y, Nakai A, Namula Z, Nagahara M, Tanihara F, Hirata M, and Otoi T (2024) Efficient gene editing of pig embryos by combining electroporation and lipofection, Veterinary World, 17(11): 2701-2707.

Received: 2024-09-10 Accepted: 2024-10-29 Published online: 2024-11-30

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

DOI: 10.14202/vetworld.2024.2701-2707

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.