Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system

• Published in: Veterinary and animal science Vol. 16; p. 100241
• Main Authors: Namula, Zhao, Wittayarat, Manita, Do, Lanh Thi Kim, Van Nguyen, Thanh, Lin, Qingyi, Takebayashi, Koki, Hirata, Maki, Tanihara, Fuminori, Otoi, Takeshige
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.06.2022; Elsevier
• Subjects: CRISPR/Cas9; Electroporation; Gene knockout; Permeability; Pig; Electroporation; Permeability; CRISPR/Cas9; Gene knockout; Pig
• ISSN: 2451-943X; 2451-943X
• DOI: 10.1016/j.vas.2022.100241

•Mosaicism is a serious problem for genome editing during embryogenesis.•We hypothesized that genome-editing before in vitro fertilization can increase its efficiency.•We introduced CRISPR/Cas9 system into oocytes during in vitro maturation using electroporation.•Gene editing efficiency in matured oocytes was comparable with that in fertilized zygotes.•Matured oocytes are suggested as functional material accepting gene editing application. 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.