Skaros, A.; Vitriolo, A.; Leonardi, O.; Finazzi, V.; Pereira, M. F.; Prazzoli, F.; Trattaro, S.; Moriano, J.; Capocefalo, D.; Villa, C. E.; Boettcher, M.; Boeckx, C.; Testa, G.

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens-specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15, NOVA1, RB1CC1 and SPAG5 acting as central regulator across cortical cell types.