Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein

• Published in: International journal of molecular sciences 2022-09, Vol.23 (18), p.10678
• Main Authors: Takabayashi, Shuji, Iijima, Kenta, Tsujimura, Masumi, Aoshima, Takuya, Takagi, Hisayoshi, Aoto, Kazushi, Sato, Masahiro
• Format: Article
• Language: English
• Subjects: Acids; Communication; CRISPR; cumulus cells; early zygotes; Editing; Efficiency; Electroporation; Embryos; Females; Fetuses; Genome editing; Genomes; Hamsters; i-GONAD; in vivo electroporation; New technology; Nucleic acids; Oviduct; Pregnancy; Proteins; Reagents; Vagina; Zygotes
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms231810678

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00–13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.