Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.
Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.
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作者：
最近，II型聚集规律间隔短回文重复序列（CRISPR）系统的进展有望改善基因组编辑方法。然而，该系统在模式生物（如斑马鱼）中的适用性和效率研究甚少。在这里，我们报告了rna引导的Cas9核酸酶以一种简单而强大的方式有效地促进了哺乳动物细胞和斑马鱼胚胎的基因组编辑。在斑马鱼体内胚胎中，当使用特异性Cas/gRNA靶向etsrp、gata4或gata5时，发现超过35%的位点特异性体细胞突变。Cas9/gRNA有效地诱导了etsrp或gata5在生成的体细胞中的双等位基因转化，在etsrpy11突变胚胎中再现了它们各自的血管表型，在fautm236a突变胚胎中再现了它们各自的心裂表型。最后，我们成功实现了Cas9/gRNA系统诱导mloxP序列在斑马鱼胚胎中的位点特异性插入。这些结果表明，Cas9/gRNA系统具有成为斑马鱼和其他模式生物简单、稳健和高效的反向遗传工具的潜力。与其他基因组工程技术一起，Cas9系统有望应用于生物学、农业、环境研究和医学。
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 etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a 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.
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影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
DOI：
10.1126/science.1192272
发表时间：
2010-09-10
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Haurwitz RE;Jinek M;Wiedenheft B;Zhou K;Doudna JA
通讯作者：
Doudna JA