Human Accelerated Regions regulate gene networks implicated in apical-to-basal neural progenitor fate transitions

The evolution of the human cerebral cortex involved modifications in the composition and proliferative potential of the neural stem cell (NSC) niche during brain development. Genetic changes that altered the activity of transcriptional enhancers have been linked to phenotypic differences between humans and other primates. Human Accelerated Regions (HARs) consist of >1600 highly constrained noncoding regions of the genome that show significant evolutionary acceleration on the human lineage, suggesting they encode human-specific functions. Multiple studies support that HARs include neurodevelopmental enhancers with novel activities in humans, but the regulatory roles of HARs in NSC biology has not been empirically assessed at scale. Here we conducted a direct-capture Perturb-seq screen repressing 180 neurodevelopmentally active HARs in human iPSC-derived NSCs with single-cell transcriptional readout. After profiling over 188,000 NSCs, we identified a set of HAR perturbations with convergent transcriptional effects on gene networks related to the specification of basal radial glia (bRG), a progenitor population that is expanded in humans. Processes associated with these networks included the regulation of apicobasal polarity and migration. We found convergent dysregulation of specific apicobasal polarity and adherens junction regulators, including PARD3, ABI2, SETD2, and PCM1, which are critical to apical-basal RG fate decisions. Autism- and intellectual-disability-associated genes were also enriched among genes showing changes in expression due to HAR perturbations. Our findings reveal interconnected roles for HARs in NSC biology and cortical development and link specific HARs to processes implicated in human cortical expansion. Polyclonal human iPSC cells (line H23555, a gift from Y. Gilad) expressing KRAB:dCas9 were inducted into neural stem cells via dual SMAD inhibition for 3 days, then transduced with a direct-capture sgRNA library targeting 180 Human Accelerated Regions, 5 positive control loci, and 6 non-targeting control sequences. Cells were recovered then sorted for lentiviral BFP, and neural induction continued to 9 days. Experiments were performed in 3 batches across 8 GEM lanes each.