Massively parallel reporter assay investigates shared genetic variants of eight psychiatric disorders

A meta-genome-wide association study across eight psychiatric disorders has highlighted the genetic architecture of pleiotropy in major psychiatric disorders. However, mechanisms underlying pleiotropic effects of the associated variants remain to be explored. We conducted a massively parallel reporter assay to decode the regulatory logic of variants with pleiotropic and disorder-specific effects. Pleiotropic variants differ from disorder-specific variants by exhibiting chromatin accessibility that extends across diverse cell types in the neuronal lineage and by altering motifs for transcription factors with higher connectivity in protein-protein interaction networks. We mapped pleiotropic and disorder-specific variants to putative target genes using functional genomics approaches. In vitro CRISPR perturbation of selected variants confirmed the variant-gene relationships, and in vivo CRISPR perturbation of a pleiotropic and a disorder-specific gene suggests that pleiotropy may involve regulation of genes expressed broadly across neuronal cell types and with higher network connectivity. To explore molecular mechanisms underlying pleiotropy, we conducted CROP-seq (CRISPR knockout followed by single-cell RNA sequencing) in the mouse neocortex on two genes: Anp32e (mapped to a pleiotropic locus), and Kmt5a (mapped to a disease-specifc locus). CRISPR guide-RNAs (gRNAs) targeting Anp32e and Kmt5a were expressed via CROPseq-tRFP lentiviral library in neonatal Cas9 transgenic mice. At two weeks of age, single cells from the neocortex were dissociated, sorted for RFP expression, then procssed with 10x Genomics 3' workflow for construction of single-cell gene expression and gRNA libraries. The high-throughput sequencing (HTS) data from the gene and gRNA libraries were used for downstream analysis (cell type annotation, differential gene expression analysis, etc).