{"doi":"10.1101/2023.03.24.534001","title":"split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":398855,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9491,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":300182,"name":"Haojiang Luan","orcid":null,"position":1,"is_corresponding":false},{"id":648170,"name":"Jun Xu","orcid":"0000-0002-7963-0253","position":2,"is_corresponding":false},{"id":623221,"name":"Rohit Singh","orcid":"0000-0002-4084-7340","position":3,"is_corresponding":false},{"id":714336,"name":"Neha Joshi","orcid":"0000-0001-5685-4927","position":4,"is_corresponding":false},{"id":892275,"name":"Tanuj Thakkar","orcid":null,"position":5,"is_corresponding":false},{"id":3585,"name":"Bonnie Berger","orcid":"0000-0002-2724-7228","position":6,"is_corresponding":false},{"id":298433,"name":"Benjamin H. White","orcid":"0000-0003-0612-8075","position":7,"is_corresponding":false},{"id":14957,"name":"Norbert Perrimon","orcid":"0000-0001-7542-472X","position":8,"is_corresponding":false},{"id":259034,"name":"Ben Ewen‐Campen","orcid":"0000-0002-4326-3033","position":0,"is_corresponding":true}],"reference_count":52,"raw_metadata":null,"created_at":"2026-07-19T01:19:51.724387Z","pmid":"36993523","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}