The sparse driver system forin vivosingle-cell labeling and manipulation inDrosophila

SUMMARY In this protocol, we introduce a sparse driver system for cell-type specific single-cell labeling and manipulation in Drosophila , enabling complete and simultaneous expression of multiple transgenes in the same cells. The system precisely controls expression probability and sparsity via mutant FRT sites with reduced recombination efficiency and tunable FLP levels adjusted by heat-shock durations. We demonstrate that this generalizable toolkit enables tunable sparsity, multi-color staining, single-cell trans-synaptic tracing, single-cell manipulation, and in vivo analysis of cell-autonomous gene function. For details on the use and execution of this protocol, please refer to Xu et al. 2024. GRAPHICAL ABSTRACT