Cerebellar malformations, such as cerebellar hypoplasia, are associated with genes relating to neurodevelopmental disorders (NDDs); however, the cellular mechanisms driving cerebellar hypoplasia are largely unknown. The Autism Susceptibility Candidate 2 (AUTS2) gene is one such gene implicated in cerebellar hypoplasia. Interestingly, patients with severe cerebellar hypoplasia typically have mutations within exon 9 of AUTS2. AUTS2 has been studied in cerebral organoid models, but not in cerebellar models (where hypoplasia occurs in patients). 

Here, we establish cell lines and generate cerebellar organoids to study AUTS2. Using CRISPR-Cas9 targeting exon 9 of AUTS2, we generated a mosaic pool of AUTS2 mutant cells and isolated subclones for PCR and Sanger sequencing analysis. Among 26 screened clones, we isolated two homozygous frameshift mutant lines (-1 and +1 bp) and one heterozygous frameshift (-5 bp) mutant line. Additionally, western blotting revealed the baseline expression of AUTS2 protein in WT stem cells, Ngn2 induced cortical neurons, and cerebellar organoids. AUTS2 is minimally expressed in undifferentiated stem cells but is robustly expressed as two isoforms in Ngn2 neurons and mature cerebellar organoids. Both Ngn2 neurons and cerebellar organoids express a long AUTS2 isoform (~130 kDa). However, cerebellar organoids express a smaller short isoform (~70 kDa) compared to the short isoform observed in Ngn2 neurons (~100 kDa). Finally, we have generated cerebellar organoids to compare the effects between homozygous and heterozygous exon 9 mutations. Future work will include immunostaining and developmental size analysis to characterize these mutant lines and determine how AUTS2 mutations affect early human cerebellar development.