Generation of genome-modified Drosophila cell lines using SwAP

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal <i>Drosophila</i> cell lines has remained a longstanding challenge, hampered by the difficulty of getting <i>Drosophila</i> cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered <i>Drosophila</i> cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited <i>armadillo</i> mutant <i>Drosophila</i> cell lines. Our method provides a powerful and simple workflow that improves the utility of <i>Drosophila</i> cells for genetic studies with CRISPR/Cas9.