Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.
Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.
复制标题
利用转基因 Cas9 在果蝇中进行高效基因敲除和敲入
DOI：
10.1534/g3.114.010496
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发表时间：
2014-03-21
期刊：
影响因子：
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通讯作者：
Gao G
中科院分区：
文献类型：
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作者：
Xue Z;Ren M;Wu M;Dai J;Rong YS;Gao G
细菌Cas9核酸酶利用小的引导RNA（gRNA）作为向导诱导位点特异性DNA断裂。Cas9已成功引入果蝇用于基因组编辑。在此，我们通过开发一种在果蝇生殖系中表达Cas9的转基因系统来提高该方法的通用性。利用这一系统，我们仅将gRNA注入胚胎就诱导出可遗传的敲除突变，通过从一个新的非编码RNA基因的启动子表达gRNA实现了高效诱变，并且通过将gRNA与环状DNA供体共同注入，以4.5%的比率实现了基于同源重组的荧光标记基因敲入。
Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.