Institute of Molecular Medicine, Peking University, Beijing 100871, China.
Cell Res. 2013 Apr;23(4):465-72. doi: 10.1038/cr.2013.45. Epub 2013 Mar 26.
Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp(y11) mutant embryos or cardia bifida phenotypes in fau(tm236a) mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.
近年来，II 型簇状规律间隔短回文重复（CRISPR）系统的进展有望为基因组编辑提供一种改进的方法。然而，该系统在斑马鱼等模式生物中的适用性和效率还鲜有研究。在这里，我们报告 RNA 引导的 Cas9 核酸酶以简单而稳健的方式有效地促进了哺乳动物细胞和斑马鱼胚胎中的基因组编辑。当特定的 Cas/gRNA 用于靶向斑马鱼胚胎中的 etsrp、gata4 或 gata5 时，发现超过 35%的特定位点的体细胞突变。Cas9/gRNA 有效地诱导了 etsrp 或 gata5 在所得体细胞中的双等位基因转换，重现了它们在 etsrp(y11)突变胚胎中的血管表型或在 fau(tm236a)突变胚胎中的心脏二分畸形表型。最后，我们成功地在斑马鱼胚胎中实现了由 Cas9/gRNA 系统诱导的 mloxP 序列的特异性插入。这些结果表明，Cas9/gRNA 系统有可能成为斑马鱼和其他模式生物的一种简单、稳健和高效的反向遗传学工具。与其他基因组工程技术一起，Cas9 系统有望在生物学、农业、环境研究和医学中得到应用。
