Ian R. Jones

Abstract Advances in genomics have led to the identification of many risk loci with hundreds of genes and thousands of DNA variants associated with neuropsychiatric disorders. A significant barrier to understanding the genetic underpinnings of complex diseases is the lack of functional characterization of risk genes and variants in biological systems relevant to human health and connecting disease-associated variants to pathological phenotypes. Characterizing gene and DNA variant functions requires genetic perturbations followed by molecular and cellular assays of neurobiological phenotypes. However, generating null or mutant alleles is low throughput, making it impossible to characterize disease-associated variants in large quantities efficiently. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens can be leveraged to dissect the biological consequences of the tested genes and variants in their native context. Nevertheless, testing non-coding variants associated with complex diseases remains non-trivial. In this review, we first discuss the current challenges of interpreting the function of the non-coding genome and approaches to prioritizing disease-associated variants in the context of the 3D epigenome. Second, we provide a brief overview of high-throughput CRISPRi and CRISPRa screening strategies applicable for characterizing non-coding sequences in appropriate biological systems. Lastly, we discuss the promising prospects of using CRISPR-based technologies to dissect DNA sequences associated with neuropsychiatric diseases.

Abstract Genome-wide association studies (GWAS) have identified thousands of non-coding variants that contribute to psychiatric disease risks, likely by perturbing cis -regulatory elements (CREs). However, our ability to interpret and explore their mechanisms of action is hampered by a lack of annotation of functional CREs (fCREs) in neural cell types. Here, through genome-scale CRISPR screens of 22,000 candidate CREs (cCREs) in human induced pluripotent stem cells (iPSCs) undergoing differentiation to excitatory neurons, we identify 2,847 and 5,540 fCREs essential for iPSC fitness and neuronal differentiation, respectively. These fCREs display dynamic epigenomic features and exhibit increased numbers and genomic spans of chromatin interactions following terminal neuronal differentiation. Furthermore, fCREs essential for neuronal differentiation show significantly greater enrichment of genetic heritability for neurodevelopmental diseases including schizophrenia (SCZ), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD) than cCREs. Using high-throughput prime editing screens we experimentally confirm 45 SCZ risk variants that act by affecting the function of fCREs. The extensive and in-depth functional annotation of cCREs in neuronal types therefore provides a crucial resource for interpreting non-coding risk variants of neuropsychiatric disorders.

Abstract The human cortex is complex and heterogeneous, undergoing extensive expansion during development. Our study of neurogenesis, including radial glia (RG), intermediate progenitor cells (IPCs), excitatory neurons (eNs), and interneurons (iN) demonstrated that chromatin looping underlies transcriptional regulation for lineage-specific genes, shedding insight on how non-coding genetic variants contribute to neuropsychiatric disorders via cell type-specific gene regulation. Radial glia play a crucial role in generating cellular diversity through both neurogenesis and gliogenesis and can be further classified into ventricular radial glia (vRG) and outer radial glia (oRG). Given their significance in cortical development, we conducted a comprehensive 3D epigenomic analysis of four major glial populations, including vRG, oRG, oligodendrocyte precursor cells (OPC), and microglia (MG), from the mid-gestational human neocortex. By integrating gene expression, chromatin accessibility, DNA methylation, and 3D chromatin interactions, we identified cell type-specific candidate cis-regulatory elements (cCREs) and validated their enhancer function using transgenic mouse embryos. Using machine learning, we prioritized 112 schizophrenia risk variants within glia cCREs and further confirmed the predicted vRG enhancer disruption by rs4449074 risk allele in vivo. Finally, oRG cCREs are enriched for human accelerated regions (HARs) compared to other cCREs and a subset of HARs have predicted activity differences compared to their chimpanzee orthologs that interact with genes involved in neuronal development. Our findings advance the understanding of human-specific gene regulation during corticogenesis.

Abstract Genome organization and the transcriptome undergo substantial changes during human brain development. However, the precise developmental dynamics of the 3D epigenome and their role in regulating transcriptomic diversity and cellular function remain poorly understood. To fill this gap, we generated and integrated 3D epigenome and transcriptome data of excitatory neurons, inhibitory neurons, oligodendrocytes, microglia, and astrocytes across second-trimester, third-trimester, neonatal, and adolescent human cortical samples. As neurons matured, we observed strengthened long-range chromatin interactions coupled with the emergence of more complex gene co-expression networks, enriched for functions related to neuronal differentiation and maturation. Furthermore, by leveraging machine learning, we identified key features predictive of cell type-specific RNA splicing, including chromatin accessibility and 3D interaction, enabling the prediction of splicing events from single-cell multi-omics data of diverse brain samples and identification of potential disease-associated splicing events. Overall, our findings illuminate how the 3D epigenome is dynamically rewired to regulate gene expression and RNA splicing during cortical development.