Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, UK.
Biol Open. 2014 Jan 15;3(1):42-9. doi: 10.1242/bio.20137120.
We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. By targeting a constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to 82% of cells, thereby allowing the study of genetic knockouts in a Drosophila cell line for the first time. We have shown that homologous gene targeting is possible at 1-4% efficiency using this system, allowing for the construction of defined insertions and deletions. We demonstrate that a 1 kb homology arm length is optimal for integration by homologous gene targeting, and demonstrate its efficacy by tagging the endogenous AGO1 protein. This technology enables controlled genetic manipulation in Drosophila cell lines, and its simplicity offers the opportunity to study cellular phenotypes genome-wide.
我们已经将 CRISPR/Cas9 系统应用于果蝇 S2 细胞，以在超过 85%的等位基因中产生靶向遗传突变。通过靶向 AGO1 基因的一个组成型外显子，我们证明了高达 82%的细胞发生纯合突变，从而首次允许在果蝇细胞系中研究基因敲除。我们已经表明，使用该系统可以以 1-4%的效率实现同源基因靶向，从而允许构建定义的插入和缺失。我们证明，同源基因靶向的最佳整合 1 kb 同源臂长度，并通过标记内源性 AGO1 蛋白证明其功效。这项技术使 Drosophila 细胞系中的受控遗传操作成为可能，其简单性为研究全基因组细胞表型提供了机会。
