Vet World Vol.15 September-2022 Article-12
Research Article
Veterinary World, 15(9): 2210-2216
https://doi.org/10.14202/vetworld.2022.2210-2216
Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods
2. Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan.
3. Faculty of Veterinary Science, Prince of Songkla University, 90110 Songkhla, Thailand.
4. Department of Veterinary Medicine, College of Coastal Agricultural Sciences, Guangdong Ocean University, 524088 Guangdong, China.
5. Department of Animal Theriogenology and Surgery, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, 100000 Hanoi, Vietnam.
Background and Aim: Mosaicism – the presence of both wild-type and mutant alleles – is a serious problem for zygotic gene modification through gene editing using the Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/ Cas9) system. Different delivery methods, such as microinjection (MI), electroporation (EP), and transfection (TF), can be used to transfer CRISPR/Cas9 components into porcine zygotes. This study aimed to develop a method that combines MI, EP, and TF to improve mutation efficiency mediated through the CRISPR/Cas9 system for a triple-gene knockout in pigs.
Materials and Methods: The study consisted of three groups: The MI group with three simultaneously microinjected guide RNAs (gRNAs) targeting α-1,3-galactosyltransferase (GGTA1), cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and β-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2); the MI + EP group with two gRNAs targeting GGTA1 and B4GALNT2 genes delivered into zygotes through MI, followed by EP of gRNA targeting the CMAH 1 h later; and the MI + EP + TF group with MI of gRNA targeting GGTA1 gene into zygotes, followed by EP of gRNA targeting CMAH 1 h later, and then TF of gRNA targeting the B4GALNT2 gene into zona-free zygotes after another hour.
Results: The rate of blastocysts carrying mutations in one or two gene(s) was significantly higher in the MI + EP + TF group than in the MI group. However, the blastocyst formation rate of zygotes in the MI + EP + TF group was lower than that of the zygotes in the other treatment groups.
Conclusion: The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts. Keywords: clustered regularly interspaced short palindromic repeats/Cas9, electroporation, microinjection, porcine zygotes, transfection.
Keywords: clustered regularly interspaced short palindromic repeats/Cas9, electroporation, microinjection, porcine zygotes, transfection.
How to cite this article: Le QA, Wittayarat M, Namula Z, Lin Q, Takebayashi K, Hirata M, Tanihara F, Do LTK, and Otoi T (2022) Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods, Veterinary World, 15(9): 2210–2216.
Received: 31-03-2022 Accepted: 17-08-2022 Published online: 16-09-2022
Corresponding author: Fuminori Tanihara E-mail: f_tanihara@jichi.ac.jp
DOI: 10.14202/vetworld.2022.2210-2216
Copyright: Le, 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.