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&emsp14;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.