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

Area of Science:

• Genomics
• Developmental Biology
• Molecular Biology

Background:

• Measuring cis-regulatory element (CRE) activity in multicellular systems at scale and with precision is a significant challenge in genomics.
• Existing methods for multiplex single-cell reporter assays face limitations in detection and quantification.
• Accurate characterization of CREs is crucial for understanding developmental processes.

Purpose of the Study:

• To develop a novel reporter assay for scalable and precise measurement of CRE activity in single cells within multicellular systems.
• To overcome the bottleneck in quantifying developmental CREs.
• To enable high-throughput screening and characterization of CREs.

Main Methods:

• Development of a dual RNA cassette system to decouple detection and quantification in reporter assays.
• Utilizing RNA barcode stabilization via circularization for enhanced reporter stability and readout.
• Application of the assay to screen over 200 accessible chromatin regions in an in vitro model of early mammalian development.
• Analysis of chimeric CRE pairs and CRE variants with perturbed transcription factor binding sites.

Main Results:

• The developed reporter assay provides accurate measurements over multiple orders of magnitude with precision near the Poisson limit.
• Identification of 13 developmental CREs, including 8 previously uncharacterized ones, with autonomous and cell-type-specific activity.
• Demonstration that chimeric CRE pairs can generate distinct activity profiles in two cell types.
• Assessment of gain- and loss-of-function phenotypes from modified CREs.

Conclusions:

• The single-cell quantitative expression reporters offer a scalable, high-sensitivity, and high-resolution method for characterizing native, perturbed, and synthetic CREs.
• This technology significantly advances the ability to study CRE function in developmental and multicellular contexts.
• The findings provide new insights into the regulatory mechanisms governing early mammalian development.