Regulatory logic of human cortex evolution by combinatorial perturbations

Abstract

5 10 15 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. Main Text 20 25 30 35 40 45 The availability of high-coverage genomes from our closest extinct relatives (1-4) offers the unprecedented opportunity to probe at high resolution the biological foundations underlying the distinctively modern human condition (5-7). Among such differences we expect to find changes impacting early brain development, as the fossil record points to a distinct brain ontogeny reflected in the distinctive contemporary sapiens globular neurocranium (8). As is to be expected from a highly polygenic trait, bioinformatic analyses have provided a long list of plausible variants of interest (9), but the precise impact of many of these still await functional validation. Brain organoids appear to be ideally-suited experimental models to capture the nature of early brain growth differences, as they recapitulate fetal brain development with a high degree of fidelity (10). Indeed, attempts have been made to interrogate the impact of derived single nucleotide variants by growing brain organoids containing the CRISPR-engineered ancestral variant of interest and comparing them with typically developing human brain organoids (11-13). Until now studies have mostly focused on understanding the impact of sapiens-derived alleles in protein-coding regions that are virtually fixed across modern populations compared to the genomes of extinct hominins. Such work has offered valuable insights, but remains limited in several respects. …