Lentiviral single-cell MPRA of synthetic enhancers reveals motif affinity-based encoding of cell state specificity [sc-lentiMPRA]

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.

Here, we developed 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 profiled 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.