Influence of Electroporation Timing on CRISPR/Cas-Mediated Multiple Gene Editing in Buffalo Embryos

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Gene editing in large animals like buffalo is challenged by mosaicism, where cells contain both wild-type and mutant alleles, complicating the creation of genetically modified F0 animals in a single step. Traditionally, electroporation is performed on zygotes post in vitro fertilization (IVF), but mature oocytes’ higher permeability suggests earlier intervention might reduce mosaicism and enhance editing efficiency. We hypothesized that the timing of electroporation before 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, GDF9, and POU5F1 were simultaneously introduced into the oocytes that had been incubated for 44, 46, and 48 h from the start of the IVM culture. Electroporation with three gRNAs at 44 h and 46 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 48 h of IVM culture were similar to those of control zygotes electroporated at 12 h after the initiation of IVF. In conclusion, multiple gene editing efficiency in the resulting blastocysts was comparable between oocytes electroporated before and after fertilization, indicating that oocytes with completed maturation time may allow better functioning of materials accepting gene editing application.