Massively parallel reporter assay for mapping gene-specific regulatory regions at single-nucleotide resolution.

Precise gene regulation is essential for tissue development and function, yet mapping cis-regulatory modules (CRMs) at high resolution and in specific cell types remains challenging. We introduce two complementary strategies-a locus-specific massively parallel reporter assay (LS-MPRA) and a degenerate MPRA (d-MPRA)-designed to overcome limitations in throughput, resolution, and prior knowledge requirements. LS-MPRA uses BAC-based libraries to densely sample genomic regions, enabling unbiased interrogation of millions of DNA fragments for CRM activity. D-MPRA applies systematic mutagenesis to resolve CRM architecture at single-nucleotide resolution, nominating essential bases that may function as TF binding sites or other regulatory elements. We applied these methods to retinal genes expressed in mature rods and bipolar interneurons using in vivo and ex vivo mouse (Mus musculus) tissue. LS-MPRA recapitulated known CRMs and identified previously uncharacterized CRMs, including those embedded in neighboring genes. Applied to Olig2, a dynamically expressed gene in retinal progenitors, LS-MPRA identified three CRM regions, which d-MPRA and motif analyses further dissected. CUT&RUN confirmed direct binding of candidate TFs. Extending LS-MPRA to chick (Gallus gallus) retina and spinal cord demonstrated cross-species and cross-tissue applicability. Together, these approaches provide a rapid, scalable, inexpensive, and accessible platform for CRM discovery that can be carried out without prior element annotation and with tunable (small) fragment sizes.