Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

Summary Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene-fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome-editing have been established in only a small set of animals. To overcome these issues, we developed a method of large fragment DNA knock-in without micromanipulation. In this study, we successfully delivered the knock-in donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large DNA fragment knock-in and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.