Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila
Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila
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DOI：
10.1534/genetics.113.156737
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发表时间：
2013-11-01
期刊：
影响因子：
3.3
通讯作者：
Ueda, Ryu
中科院分区：
文献类型：
--
作者：
Kondo, Shu;Ueda, Ryu
我们报告了一个简单但极其有效的平台，用于在果蝇中通过 RNA 引导的核酸内切酶 Cas9 进行系统基因靶向。该系统包含两种转基因菌株：一种从种系特异性 nanos 启动子表达 Cas9 蛋白，另一种普遍表达针对基因组中独特位点的定制引导 RNA (gRNA)。这两种菌株杂交后会在生殖细胞中形成活性 Cas9-gRNA 复合物，从而切割和突变目标位点。我们证明了七个神经肽和两个 microRNA 基因中突变体的快速生成，其中尚未描述突变体。稳定表达 Cas9-gRNA 的创始动物将种系突变传递给平均 60% 的后代，与之前基于瞬时 Cas9 表达的方法相比，效率有了显着提高。通过共表达两个 gRNA 来同时切割两个位点可有效诱导内部缺失，频率为 4.3-23%。我们的方法很容易扩展到高通量基因靶向，从而加速果蝇基因组的全面功能注释。
We report a simple yet extremely efficient platform for systematic gene targeting by the RNA-guided endonuclease Cas9 in Drosophila. The system comprises two transgenic strains: one expressing Cas9 protein from the germline-specific nanos promoter and the other ubiquitously expressing a custom guide RNA (gRNA) that targets a unique site in the genome. The two strains are crossed to form an active Cas9-gRNA complex specifically in germ cells, which cleaves and mutates the target site. We demonstrate rapid generation of mutants in seven neuropeptide and two microRNA genes in which no mutants have been described. Founder animals stably expressing Cas9-gRNA transmitted germline mutations to an average of 60% of their progeny, a dramatic improvement in efficiency over the previous methods based on transient Cas9 expression. Simultaneous cleavage of two sites by co-expression of two gRNAs efficiently induced internal deletion with frequencies of 4.3-23%. Our method is readily scalable to high-throughput gene targeting, thereby accelerating comprehensive functional annotation of the Drosophila genome.