Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector
Naoaki Mizuno,
Eiji Mizutani,
Hideyuki Sato,
Mariko Kasai,
Aki Ogawa,
Fabian Suchy,
Tomoyuki Yamaguchi,
Hiromitsu Nakauchi
Affiliations
Naoaki Mizuno
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan
Eiji Mizutani
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan
Hideyuki Sato
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan
Mariko Kasai
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan
Aki Ogawa
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan
Fabian Suchy
Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
Tomoyuki Yamaguchi
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan; Corresponding author
Hiromitsu Nakauchi
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 1088639, Japan; Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Corresponding author
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 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. : Techniques in Genetics; Genetic Engineering Subject Areas: Techniques in Genetics, Genetic Engineering
