Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts

Abstract

Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).

Keywords: CRISPR/Cas; Electroporation; Embryos; Genome editing; Mouse.