Micro-exons are exons, 3-27nt in length, that modulate protein-protein interactions and are specifically spliced-in in neurons, affecting mRNAs involved in neuronal development and synaptic function. Differential inclusion of micro-exons has been reported in post-mortem brains of individuals diagnosed with autism spectrum disorder (ASD). This was attributed to a reduced expression of the splicing factor SRRM4. However, when, during development, mis-splicing occurs in ASD and which cell types are affected remains unclear. Brain organoids, derived from human embryonic or induced pluripotent stem cells, recapitulate early human brain development to a large extent and can therefore be used to elucidate the extent and impacts of dysregulated micro-exon splicing. In support of this idea, our bioinformatic analyses of in-house brain organoid models of neurodevelopmental disorders identified consistent patterns across several conditions, including Down Syndrome (DS), Ataxia-Telangiectasia (AT), and Hypomyelination with Brainstem and Spinal cord involvement and Leg Spasticity (HBSL). To systematically investigate the role of micro-exon splicing we utilized the CRISPR-Cas9 system to knockout (KO) the micro-exon splicing factors SRRM4 and its paralog, SRRM3, in H9 embryonic stem cells and established clonally derived lines with validated frameshift mutations. We are currently utilizing these lines and their isogenic controls to generate cortical brain organoids that will be subjected to full-length single-cell RNA sequencing (scRNA-seq) across multiple timepoints during brain organoid maturation. This systematic analysis should unravel how disruptions in SRRM4 and SRRM3 function are associated with the pathophysiology of neurodevelopmental disorders and elucidate which mRNA species are affected in each cell type across development.