Abstract The split-Gal4 system allows for intersectional genetic labeling of highly specific cell-types and tissues in Drosophila . However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a new split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is fully repressible by Gal80. We demonstrate the potent inducibility of “split-intein Gal4” in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell-type specific genetic drivers based on in silico predictions generated by single cell RNAseq (scRNAseq) datasets, and we describe a new algorithm (“Two Against Background” or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible. Significance statement The split-Gal4 system allows Drosophila researchers to drive transgene expression with extraordinary cell type specificity. However, the existing split-Gal4 system cannot be controlled temporally, and therefore cannot be applied to many important areas of research. Here, we present a new split-Gal4 system based on a self-excising split-intein, which is fully controllable by Gal80, as well as a related drug-inducible split GeneSwitch system. This approach can both leverage and inform single-cell RNAseq datasets, and we introduce an algorithm to identify pairs of genes that precisely and narrowly mark a desired cell cluster. Our split-intein Gal4 system will be of value to the Drosophila research community, and allow for the creation of highly specific genetic drivers that are also inducible/repressible.

split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80