High-Throughput Functional Annotation of Ultra-Rare Schizophrenia Risk Variants through CRISPR Knockout Screening
1 Cellular and Gene Editing Research, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UKu200b
2 Institute of Human Genetics, University Hospital Bonn, Germany
3 Human Genetics, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK u200b
4 Department of Psychiatry and Psychotherapy University Hospital Bonnu200b, Germany
5 Human Genetics, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK
6 Institute of Psychiatric Phenomics and Genomics (IPPG), University of Munich, Germany u200b
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, employing dual guide RNAs and three independent approaches per gene to achieve precise and robust knockouts in a pooled format. Pooled CRISPR-Cas9 libraries targeting all genes of interest were used to infect iPSCs (KOLF 2.1S) that were subsequently differentiated into glutamatergic iNeurons. 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. SynGo analysis showed alterations in both pre- and post-synaptic compartments. Context-dependent analyses are currently ongoing. By conducting 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.