CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice
Naoaki Mizuno,
Eiji Mizutani,
Hideyuki Sato,
Mariko Kasai,
Hiromitsu Nakauchi,
Tomoyuki Yamaguchi
Affiliations
Naoaki Mizuno
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Eiji Mizutani
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Hideyuki Sato
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Mariko Kasai
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Hiromitsu Nakauchi
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, JapanInstitute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, California, USA
Tomoyuki Yamaguchi
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos’ genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.
