A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes

CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.