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

• DOI: 10.1016/j.vas.2022.100241
• Publication Date: 2022-02-28T17:11:20Z
• Authors: Zhao Namula; Manita Wittayarat; Lanh Thi Kim Do; Thanh Van Nguyen; Qingyi Lin; Koki Takebayashi; Maki Hirata; Fuminori Tanihara; Takeshige Otoi
• Keywords: Gene knockout

Mosaicism, including alleles comprising both wild-type and mutant, is a serious problem for gene modification by editing using electroporation. One-step generation of F0 pigs with completely desired modifications saves cost time, but the major obstacles have been mosaic mutations. We hypothesized that timing electroporation prior to in vitro fertilization (IVF) can increase rates biallelic mutation multiple knockout as permeability mature oocytes greater than zygotes. Hence, we determined whether during maturation (IVM) culture enhances triple resulting blastocysts. Three gRNAs targeting KDR, PDX1, SALL1 were simultaneously introduced into had incubated 40, 42, 44 h from start IVM culture. Electroporation three at 40 42 decreased blastocyst formation did not improve target number mutations The rate, rates, numbers blastocysts treated similar those control zygotes electroporated 13 after initiation IVF. In conclusion, efficiency was comparable between before fertilization, indicating completed time may allow better functioning materials accepting application.