Reconstructing signaling history of single cells with imaging-based molecular recording.

The intensity and duration of biological signals encode information that allows a few pathways to regulate a wide array of cellular behaviors. Despite the central importance of signaling in biomedical research, our ability to quantify it in individual cells over time remains limited. Here, we introduce INSCRIBE, an approach for reconstructing signaling history in single cells using endpoint fluorescence images. By regulating a CRISPR base editor, INSCRIBE generates mutations in genomic target sequences, at a rate proportional to signaling activity. The number of edits is then recovered through a novel ratiometric readout strategy, from images of two fluorescence channels. We engineered human cell lines for recording WNT and BMP pathway activity, and demonstrated that INSCRIBE faithfully recovers both the intensity and duration of signaling. Further, we used INSCRIBE to study the variability of cellular response to WNT and BMP stimulation, and test whether the magnitude of response is a stable, heritable trait. We found a persistent memory in the BMP pathway. Progeny of cells with higher BMP response levels are likely to respond more strongly to a second BMP stimulation, up to 3 weeks later. Together, our results establish a scalable platform for genetic recording and