ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here we build on previous knowledge to develop a simple AAV-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This Expression by Boolean Exclusion (ExBoX) system encodes for a gene of interest which is turned ON by a particular recombinase (Cre or FlpO) and turned OFF by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters both in vitro and in vivo, and further demonstrate the powerful adaptability of the system by achieving expression of a variety of virally-delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of organisms and systems.