Julia Rühle, Robert Frömel, Aina Bernal Martinez, Chelsea Szu-Tu, Joseph Bowness et al.
Background: Cell state-specific gene expression programs emerge from the interplay between cis-regulatory elements (CREs), such as enhancers, and transcription factors (TFs). Massively parallel reporter assays (MPRAs) have enabled large-scale dissection of CRE function, but bulk approaches cannot resolve cell state specificity on continuous trajectories of cellular differentiation, and existing single-cell MPRAs are not readily applicable to primary cell differentiation models. Results: Here, we develop a single-cell lentiviral Massively Parallel Reporter Assay (sc-lentiMPRA) that overcomes these limitations and enables parallel quantification of enhancer activity and cellular transcriptome. Applying sc-lentiMPRA in blood stem differentiation, we profile the activity and specificity of ~160 fully synthetic enhancers with controlled motif composition and affinities across ~190,000 single cells. Focusing on Trp53 and Cebpa, we show that enhancers with high and low affinity motifs differ qualitatively and quantitatively in their responses to TF expression gradients. For Trp53, low-affinity motifs exhibit near-linear correlation with TF expression, whereas high-affinity motifs show reduced sensitivity to TF levels. In contrast, Cebpa-associated enhancers display non-linear behaviors. Conclusions: Together, sc-lentiMPRA establishes a powerful framework for systematically relating enhancer architecture and TF expression to regulatory output at single-cell resolution during cellular differentiation.
Julia Rühle, Robert Frömel, Aina Bernal Martinez, Chelsea Szu-Tu, Joseph Bowness, Lars Velten. Lentiviral single-cell MPRA of synthetic enhancers reveals motif affinity-based encoding of cell state specificity. 2026-07-14. https://doi.org/10.1186/s13059-026-04186-9
Library Beta tested Cebpa motif number and affinity across synthetic enhancer designs, with random-DNA and strong-promoter controls, in primary mouse HSPCs spanning stem-cell expansion and differentiation states. The table retains construct/cell barcode observations with source design annotations and final cell-state labels.
Bulk validation lentiMPRA was performed on two biological replicates of FACS-sorted CD55-positive primary mouse HSPCs using the Alpha synthetic enhancer library. The table reports replicate RNA/DNA counts, normalized counts, and mean log2 RNA/DNA activity.
The proof-of-concept library of synthetic, endogenous, and control CREs was delivered by lentivirus to dCas9-BFP-KRAB K562 cells. The table contains enhancer-positive single-cell observations with presence-barcode and GFP quantification-barcode UMIs.
The proof-of-concept library was delivered by lentivirus to primary mouse hematopoietic stem/progenitor cells and measured during ex vivo culture. The table contains enhancer-positive single-cell observations and barcode UMI activity values.
Library Alpha tested 84 synthetic enhancer designs containing motifs for seven hematopoietic TFs, selected TF pairs, and random-background controls in primary mouse HSPCs undergoing pan-myeloid differentiation. The table retains enhancer-positive barcode/cell observations and maps final cell-state labels where the integrated Seurat metadata matches.
Bulk validation lentiMPRA was performed on two biological replicates of FACS-sorted CD55-negative primary mouse HSPCs using the Alpha synthetic enhancer library. The table reports replicate RNA/DNA counts, normalized counts, and mean log2 RNA/DNA activity.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 13 files (ZIP)13059_2026_4186_MOESM4_ESM.xlsxbulk_hsc_cd55neg_libAlpha.rdsbulk_hsc_cd55pos_libAlpha.rdsbulk_meta_libAlpha.rdsfigshare_article_31420724.jsonintegrated_seurat.rdslibAlpha_matched_gfp_guide_enhancer_counts.rdslibBeta_matched_gfp_guide_enhancer_counts.rdslibPoC_hsc_gfp_guide_enhancer_counts.rdslibPoC_hsc_seurat.rdslibPoC_k562_gfp_guide_enhancer_counts.rdslibPoC_k562_seurat.rdsREADME.txt