A microscopy reporter for cGAMP reveals rare cGAS activation following DNA damage, and a lack of correlation with micronuclear cGAS enrichment

Summary Cyclic GMP-AMP (cGAMP) synthase (cGAS) is the primary intracellular responder to pathogen DNA. Upon DNA-binding, cGAS generates cGAMP, which binds to STING, ultimately driving inflammatory signalling. Although normally silenced on self-DNA, cGAS can be activated during genotoxic stress. A universal by-product of these conditions are micronuclei, which accumulate cGAS, and which are therefore thought to be major cGAS activators. However, due to the inability to visualise cGAS activation in single cells, this hypothesis remains largely untested. Here we solve this question with an improved intracellular cGAMP reporter, which is compatible with microscopy, flow-cytometry and plate reader setups. Surprisingly, cGAS activation in response to multiple types of genotoxic stress is limited to a subfraction of cells and does not correlate with cGAS enrichment in micronuclei. Overall, our findings suggest a revised model of innate immune signalling in response to genotoxic stress, and introduce a novel and flexible tool with which to examine this model in future.