W89. HIGH-THROUGHPUT FUNCTIONAL ANNOTATION OF ULTRA-RARE SCHIZOPHRENIA RISK VARIANTS THROUGH CRISPR KNOCKOUT SCREENING

• Published in: European neuropsychopharmacology 2024-10, Vol.87, p.149-149
• Main Authors: Dominicus, Caia, Fischer, Lea, Cooper, Sarah, Feng, Claudia, Gouda, Mahesh, Salazar, Melissa, Schulze, Thomas G., Trynka, Gosia, Parts, Leopold, Bassett, Andrew, Schulte, Eva
• Format: Article
• Language: English
• ISSN: 0924-977X
• DOI: 10.1016/j.euroneuro.2024.08.298

Schizophrenia (SCZ) is a complex psychiatric disorder affecting approximately 1% of the world's population. It is associated with numerous genetic risk factors spanning the entire frequency spectrum. Among these are ultra-rare variants, which generally exhibit large effect sizes and thus, can be leveraged to understand the yet only incompletely understood pathophysiology of this condition. To bridge the gap between variant identification and functional annotation, we leveraged dual guide RNA CRISPR knockout screens in iPSC-derived excitatory neurons to elucidate the functional consequences of sixteen genes, associated with heightened SCZ risk from a whole exome sequencing-based rare variant association study (Singh T et al, Nature, 2022). Sixteen genes with an exome-wide significant enrichment of predicted loss-of-function SCZ variants were systematically targeted using a lentiviral CRISPR-Cas9 system. Dual guide RNAs and three independent approaches per gene were employed in order to achieve precise and robust knock-outs. Pooled CRISPR-Cas9 libraries targeting all genes of interest were used to infect iPSCs (KOLF 2.1S) harboring doxycycline-inducible NGN2 that were subsequently differentiated into glutamatergic iNeurons using standard protocols. Single-cell RNA sequencing (scRNAseq) was performed on the 10x Genomics plus Illumina platform. In a pilot screen, transcriptomic profiles were obtained for a total of 3000 cells, with 35 to 370 single-cell transcriptomes available per single-gene knock-out. KEGG-based preliminary pathway enrichment analyses suggest potential alterations in synaptic signaling pathways, well in line with known SCZ pathophysiology. SynGo analysis showed alterations in both pre- and post-synaptic compartments. Context-dependent analyses are currently ongoing. By high-throughput CRISPR screens, we add a first layer of function annotation in order to unravel the intricate genetic architecture of schizophrenia and its impact on critical cellular pathways. The outcomes hold significant implications for both psychiatric genetics and clinical approaches in providing an opportunity to bridge the knowledge gap between variant and gene identification and understanding underlying pathophysiology. The incomplete understanding of pathophysiology still represents the single largest hindrance to the development of precise and efficient therapeutic approaches.