Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods.

Abstract

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.

• Authors: Quynh Anh Le, Manita Wittayarat, Zhao Namula, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Lanh Thi Kim Do, Takeshige Otoi
• Journal: Veterinary world (Vet World) Vol. 15 Issue 9 Pg. 2210-2216 (Sep 2022) ISSN: 0972-8988 [Print] India
• PMID: 36341066 (Publication Type: Journal Article)
• Copyright: Copyright: © Le, et al.