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

• Published in: iScience Vol. 9; pp. 286 - 297
• Main Authors: Mizuno, Naoaki, Mizutani, Eiji, Sato, Hideyuki, Kasai, Mariko, Ogawa, Aki, Suchy, Fabian, Yamaguchi, Tomoyuki, Nakauchi, Hiromitsu
• Format: Journal Article
• Language: English
• Published: United States Elsevier 30.11.2018
• Subjects: Genetic Engineering; Techniques in Genetics
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2018.10.030

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 knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin 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.