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

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  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 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 an 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.