Cerebral Organoids Containing an AUTS2 Missense Variant

36 Variants in the AUTS2 gene are associated with a broad spectrum of neurological 37 conditions characterized by intellectual disability, microcephaly, and congenital brain 38 malformations. Here, we use a human cerebral organoid (CO) model to investigate the 39 pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a 40 patient with multiple neurological impairments including primary microcephaly and 41 profound intellectual disability. Proband COs exhibit reduced growth, deficits in neural 42 progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone43 like regions compared to control COs. We used CRISPR-Cas9-mediated gene editing to 44 correct this variant and demonstrate rescue of impaired organoid growth and NPC 45 proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S 46 transition gene expression and alterations in WNT-β-Catenin signaling within proband 47 NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. 48 Collectively, these results underscore the value of COs to uncover molecular 49 mechanisms underlying AUTS2 syndrome. 50 51