Influence of Electroporation Timing on CRISPR/Cas-Mediated Multiple Gene Editing in Buffalo Embryos
Year: 2024
Type: article
Abstract: A significant issue for gene editing via electroporation is mosaicism, which includes alleles that are both wild-type and mutant. Mosaic mutations have been the main barrier to the cost-and time-saving one-step production of F0 buffalo with all necessary gene changes. Because mature oocytes have a higher permeability than zygotes, we hypothesized that scheduling the electroporation before in vitro fertilization (IVF) could boost the rates of biallelic mutation for multiple gene deletion. Therefore, we sought to ascertain if the timing of electroporation during the culture of in vitro maturation (IVM) improves triple gene editing in the resultant blastocysts. After the oocytes had been incubated for 40, 42, and 44 hours from the beginning of the IVM culture, three gRNAs targeting KDR, GDF9, and POU5F1 were concurrently delivered. Three gRNAs were electroporated into the IVM culture at 44 and 46 hours, which reduced the rates at which blastocysts formed. However, the goal number of biallelic mutations and mutation rates in the ensuing blastocysts remained unchanged. The resultant blastocysts from oocytes treated by electroporation at 48 hours of IVM culture had equivalent blastocyst formation rates, mutation rates, and target numbers to control zygotes electroporated at 12 hours following IVF initiation. Ultimately, the efficacy of multiple gene editing in the resultant blastocysts was found to be similar in oocytes electroporated before and after fertilization. This suggests that oocytes with fully matured chromatids may have improved material properties that facilitate the application of gene editing.
Source: Veterinary Integrative Sciences
Authors: Dipankar Sardar, Md. Taslim Hossain, Rudranil Baidya, Adnan Habib
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4404531116