Single Cell Multiomics Across Nine Mammals Reveals Cell Type Specific Regulatory Conservation in the Brain

Here, we generated a multiomic data from post-mortem cortex tissue across nine mammals and identified candidate CREs (cCREs) in a cell type–specific manner. We developed a multidimensional framework of conservation, integrating sequence, chromatin accessibility, and enhancer–gene linkage. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs spanning conserved to human-specific regions. CRISPR interference (CRISPRi) validated conserved enhancer function, including at neurodevelopmental loci like FAM181B. Motif enrichment and chromBPnet revealed transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression showed both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility. RNA-seq generated from CRISPRi experiments targeting deeply conserved neuronal cCREs.