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ID 117723
Author
Namula, Zhao Guangdong Ocean University|Tokushima University
Wittayarat, Manita Prince of Songkla University
Do, Lanh Thi Kim Tokushima University|Vietnam National University of Agriculture
Nguyen, Thanh Van Tokushima University|Vietnam National University of Agriculture
Lin, Qingyi Tokushima University
Takebayashi, Koki Tokushima University
Keywords
CRISPR/Cas9
Electroporation
Gene knockout
Permeability
Pig
Content Type
Journal Article
Description
Mosaicism, including alleles comprising both wild-type and mutant, is a serious problem for gene modification by gene editing using electroporation. One-step generation of F0 pigs with completely desired gene modifications saves cost and time, but the major obstacles have been mosaic mutations. We hypothesized that the timing of electroporation prior to in vitro fertilization (IVF) can increase the rates of biallelic mutation for multiple gene knockout as the permeability of mature oocytes is greater than that of zygotes. Hence, we determined whether the timing of electroporation during in vitro maturation (IVM) culture enhances triple gene editing in the resulting blastocysts. Three gRNAs targeting KDR, PDX1, and SALL1 were simultaneously introduced into the oocytes that had been incubated for 40, 42, and 44 h from the start of the IVM culture. Electroporation with three gRNAs at 40 h and 42 h during IVM culture decreased the blastocyst formation rates and did not improve the mutation rates and target number of biallelic mutations in the resulting blastocysts. The blastocyst formation rate, mutation rates, and target numbers in the resulting blastocysts from oocytes treated by electroporation at 44 h of IVM culture were similar to those of control zygotes electroporated at 13 h after the initiation of IVF. In conclusion, multiple gene editing efficiency in the resulting blastocysts was comparable between oocytes electroporated before and after the fertilization, indicating that oocytes with completed maturation time may allow better functioning of materials accepting gene editing application.
Journal Title
Veterinary and Animal Science
ISSN
2451943X
Publisher
Elsevier
Volume
16
Start Page
100241
Published Date
2022-02-28
Rights
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
Publisher
departments
Bioscience and Bioindustry