CRISPR/Cas9与腺相关病毒介导的小鼠胚胎内基因敲除
CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice.
作者信息
Mizuno Naoaki, Mizutani Eiji, Sato Hideyuki, Kasai Mariko, Nakauchi Hiromitsu, Yamaguchi Tomoyuki
机构信息
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.
出版信息
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。
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