Mizuno Naoaki, Mizutani Eiji, Sato Hideyuki, Kasai Mariko, Ogawa Aki, Suchy Fabian, Yamaguchi Tomoyuki, Nakauchi Hiromitsu
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.
iScience. 2018 Nov 30;9:286-297. doi: 10.1016/j.isci.2018.10.030. Epub 2018 Nov 2.
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.
通过CRISPR/Cas9进行胚胎内基因组编辑，可通过非同源末端连接（NHEJ）介导的移码突变或同源定向修复（HDR）介导的点突变轻松生成基因修饰动物。然而，在没有昂贵的显微操作器的情况下，在胚胎中引入大的修饰，如基因替换或基因融合，仍然很困难。此外，仅在一小部分动物中建立了用于胚胎内基因组编辑的显微操作技术。为了克服这些问题，我们开发了一种无需显微操作的大片段DNA敲入方法。在本研究中，我们通过腺相关病毒（AAV）成功地将敲入供体DNA导入受精卵，而无需去除透明带，并且通过CRISPR/Cas9核糖核蛋白的电穿孔成功实现了大片段DNA敲入和整个外显子交换。通过这种方法，我们可以在各种动物物种中方便地进行大片段DNA交换，而无需显微操作。
