A single-cell massively parallel reporter assay detects cell-type-specific gene regulation

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Massively parallel reporter gene assays are crucial for regulatory genomics.
• Current methods require serial assays across cell types to identify cell-type-specific regulatory elements.
• This limitation hinders the comprehensive study of cis-regulatory sequences (CRSs) in diverse cellular contexts.

Purpose of the Study:

• To develop a single-cell massively parallel reporter assay (scMPRA).
• To enable simultaneous measurement of CRS activity across multiple cell types.
• To overcome the limitations of serial assays in identifying cell-type-specific regulatory elements.

Main Methods:

• Developed scMPRA for high-throughput, single-cell analysis of reporter gene activity.
• Assayed a library of core promoters in a mixture of HEK293 and K562 cells.
• Measured promoter variants across multiple cell types in live mouse retinas.

Main Results:

• scMPRA demonstrated reproducibility and high parallelism in detecting cis-regulatory activity.
• The assay successfully identified cell-type-specific cis-regulatory activity.
• Subtle genetic variants were shown to have cell-type-specific effects on CRS activity in vivo.

Conclusions:

• scMPRA is a robust single-cell reporter gene assay for dissecting cell-type-specific regulatory elements.
• The method facilitates simultaneous analysis of CRS activity across diverse cell types.
• scMPRA is anticipated to be widely applicable in studying the functional roles of CRSs in various biological systems.