Split intein vectors permit oversize FLIM-FRET biosensor neuronal expression

Adeno-associated virus (AAV) packaging limits constrain the design, performance, and in vivo application of genetically encoded biosensors. We developed a split intein-mediated reconstitution strategy enabling modular delivery and reassembly of oversized fluorescence lifetime-based biosensors. Using this platform, we engineered an oversized cAMP sensor compatible with one-photon fluorescence lifetime measurements, enabling monitoring of intracellular signaling dynamics in distinct neuronal subtypes in freely behaving mice.