Directly edit the genes of zygotes with ‘improved-Genome editing via Oviductal Nucleic Acids Delivery’The i-GONAD method delivers genome editing molecules, directly to E0.7 embryos in the oviducts of mice, via in situ electroporation. This technique generates mouse models that can contain single base changes, kilobase sized deletions and knock-ins. i-GONAD treated females retain reproductive function and are useful for generating germlines in future generations.
The University of Nebraska Medical Center’s Channabasavaiah Gurumurthy, Ph.D., collaborated with Japanese researchers Masato Ohtsuka, Ph.D., and Hiromi Miura, Ph.D., of Tokai University’s School of Medicine, invented i-GONAD . Traditionally, generating transgenic mouse-models involves three critical steps: isolation of zygotes from sacrificed females, zygote micromanipulation ex vivo and transfer of these modified zygotes into another set of female mice. This process has remained unchanged for over four decades and is laborious – requiring a high level of expertise, expensive and time-consuming.
The i-GONAD technique relives these steps by delivering genome editing nucleic acids and CRISPR components into embryos in situ. The process involves the exposure of the ovaries and oviduct of pregnant mice bearing E0.7 embryos. The genome editing reagents are injected into the oviductal lumen and the entire oviduct is subjected to electroporation using tweezer-type electrodes. These in situ, genome edited embryos are allowed to develop to term and genotyped for the targeted mutation.
This technique, when used in combination with Easi-CRISPR, another invention from Dr. Gurumurthy and Dr. Ohtsuka, inserts long single stranded DNA donor format with insertion efficiency as high as 100%. This combination changes the landscape of transgenic animal-model generation and provides a tool that is easy-to-perform and has high efficiency.
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