Mizuno Naoaki, Mizutani Eiji, Sato Hideyuki, Kasai Mariko, Nakauchi Hiromitsu, Yamaguchi Tomoyuki
Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
Bio Protoc. 2019 Jul 5;9(13):e3295. doi: 10.21769/BioProtoc.3295.
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.
利用CRISPR/Cas9进行胚胎内基因组编辑，已能够快速培育出基因敲除动物。然而，直接将大片段敲入胚胎基因组仍然困难重重，尤其是在不显微注射供体DNA的情况下。病毒载体是用于细胞系的敲入供体DNA的良好转运工具，但似乎不适用于具有透明带（围绕早期胚胎的糖蛋白膜）的植入前胚胎。我们发现腺相关病毒（AAV）能够通过完整的透明带感染各种哺乳动物的受精卵。在Cas9核糖核蛋白电穿孔后，由AAV介导的供体DNA递送能够在无需显微操作的情况下实现大片段敲入。
