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

Autor: Le QA; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan., Wittayarat M; Faculty of Veterinary Science, Prince of Songkla University, 90110 Songkhla, Thailand., Namula Z; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Department of Veterinary Medicine, College of Coastal Agricultural Sciences, Guangdong Ocean University, 524088 Guangdong, China., Lin Q; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan., Takebayashi K; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan., Hirata M; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan., Tanihara F; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan., Do LTK; Department of Animal Theriogenology and Surgery, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, 100000 Hanoi, Vietnam., Otoi T; Bio-Innovation Research Center, Tokushima University, 7793233 Tokushima, Japan.; Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 7793233 Tokushima, Japan.
Jazyk: angličtina
Zdroj: Veterinary world [Vet World] 2022 Sep; Vol. 15 (9), pp. 2210-2216. Date of Electronic Publication: 2022 Sep 16.
DOI: 10.14202/vetworld.2022.2210-2216
Abstrakt: 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.
Competing Interests: The authors declare that they have no competing interests.
(Copyright: © Le, et al.)
Databáze: MEDLINE
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