The inability to scalably and precisely measure the activity of developmental enhancers in multicellular systems is a bottleneck in genomics. Here, we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays, resulting in accurate measurement of reporter expression over a >10,000-fold range of activity with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode circularization, thesesingle-cellquantitativeexpressionreporters (scQers) provide high-contrast readouts analogous to classicin situassays, but entirely from sequencing. Screening >200 enhancers in a multicellularin vitromodel of early mammalian development, we identified numerous autonomous and cell-type-specific elements, including constituents of theSox2control region exclusively active in pluripotent cells, endoderm-specific enhancers, including nearFoxa2andGata4, and a compact pleiotropic enhancer at theLamc1locus. scQers can be mobilized in developmental systems to quantitatively characterize native, perturbed, and synthetic enhancers at scale, with high sensitivity and at single-cell resolution.
