{Reference Type}: Journal Article
{Title}: Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
{Author}: Lalanne JB;Regalado SG;Domcke S;Calderon D;Martin BK;Li X;Li T;Suiter CC;Lee C;Trapnell C;Shendure J;
{Journal}: Nat Methods
{Volume}: 21
{Issue}: 6
{Year}: 2024 Jun 9
{Factor}: 47.99
{DOI}: 10.1038/s41592-024-02260-3
{Abstract}: 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.