Total for the last 12 months
number of access : ?
number of downloads : ?
ID 117906
Author
Le, Quynh Anh Tokushima University
Wittayarat, Manita Prince of Songkla University
Namula, Zhao Tokushima University|Guangdong Ocean University
Lin, Qingyi Tokushima University
Takebayashi, Koki Tokushima University
Do, Lanh Thi Kim Vietnam National University of Agriculture
Keywords
clustered regularly interspaced short palindromic repeats/Cas9
electroporation
microinjection
porcine zygotes
transfection
Content Type
Journal Article
Description
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.
Journal Title
Veterinary World
ISSN
22310916
09728988
Publisher
Veterinary World
Volume
15
Issue
9
Start Page
2210
End Page
2216
Published Date
2022-09-16
Rights
This article is 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.
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
Publisher
departments
Bioscience and Bioindustry