Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters

作者：Jean‐Benoît Lalanne, Samuel G. Regalado, Silvia Domcke, Diego Calderon, Beth Martin, Xiaoyi Li, Tony Li, Chase C. Suiter, Choli Lee, Cole Trapnell, Jay Shendure · 发表于：Nature Methods · 年份：2024 · DOI：10.1038/s41592-024-02260-3 · 被引用次数：51 · 研究领域：Single-cell and spatial transcriptomics、RNA Research and Splicing、RNA and protein synthesis mechanisms

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) 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. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.