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 Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila 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 armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

多功能CRISPR / Cas9工具的最新开发显着提高了产生转基因动物和细胞系的难度。然而，尽管对于哺乳动物细胞系而言，等基因细胞群体的分离通常是简单的，但果蝇克隆细胞系的产生仍然是一项长期的挑战，因为果蝇很难在低密度下生长。在这里，我们描述了一种高效的工作流程，以生成由Cas9克隆的果蝇。细胞池的组合，限制在条件培养基中的稀释，并使用等位基因特异性引物进行PCR，从而能够有效选择具有合适突变谱的克隆细胞系。我们通过记录八个独立的Cas9编辑的犰狳突变果蝇细胞系的分离，选择和验证来验证该协议。我们的方法提供了强大而简单的工作流程，可改善果蝇细胞在CRISPR / Cas9基因研究中的效用。