PT  - JOURNAL ARTICLE
AU  - Jean-Benoît Lalanne
AU  - Samuel G. Regalado
AU  - Silvia Domcke
AU  - Diego Calderon
AU  - Beth Martin
AU  - Tony Li
AU  - Chase C. Suiter
AU  - Choli Lee
AU  - Cole Trapnell
AU  - Jay Shendure
TI  - Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters
AID  - 10.1101/2022.12.10.519236
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.12.10.519236
4099  - http://biorxiv.org/content/early/2022/12/10/2022.12.10.519236.short
4100  - http://biorxiv.org/content/early/2022/12/10/2022.12.10.519236.full
AB  - 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, these single-cell quantitative expression reporters (scQers) provide high-contrast readouts analogous to classic in situ assays, but entirely from sequencing. Screening >200 enhancers in a multicellular in vitro model of early mammalian development, we identified numerous autonomous and cell-type-specific elements, including constituents of the Sox2 control region exclusively active in pluripotent cells, endoderm-specific enhancers, including near Foxa2 and Gata4, and a compact pleiotropic enhancer at the Lamc1 locus. 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.Competing Interest StatementJ.S. is a scientific advisory board member, consultant and/or co-founder of Cajal Neuroscience, Guardant Health, Maze Therapeutics, Camp4 Therapeutics, Phase Genomics, Adaptive Biotechnologies, Scale Biosciences, Sixth Street Capital and Pacific Biosciences. All other authors declare no competing interests.