A massively parallel CRISPR-based screening platform for modifiers of neuronal depolarization

Mapping cell-type- and age-dependent neuronal vulnerability through genome-wide in vivo CRISPRi screens in the mouse brain.

Current brain atlases are largely descriptive, cataloging correlative molecular snapshots such as gene expression signatures yet offering limited functional insight. Here, we develop a scalable, cell-type-resolved in vivo CRISPR interference (CRISPRi) platform enabling systematic gene function profiling in the mouse brain. Through genome-wide screens across four neuronal populations at three time points spanning youth to aging, we identify neuronal essential genes missed in vitro and define a consensus set of 269 neuronal core essential genes. The data reveal cell-type-specific genetic vulnerabilities, including divergent dependencies validated for exosome component 9 (Exosc9) and osteopetrosis-associated transmembrane protein 1 (Ostm1) between excitatory and inhibitory neurons. We uncover aging-specific dependencies enriched in mitochondrial and translational pathways, aligning with transcriptional changes in the aging human brain. Finally, we establish the CRISPRinvivo data portal as a community resource for in vivo screening. Altogether, this work provides a broadly applicable platform for in vivo functional genomics and a framework for building comprehensive gene-function brain atlases.

CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder

A pooled CRISPR-based neuronal activity screen links TMEM50A-dependent MVB function to synaptic integrity and remote memory.

A high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons is established and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.

CRISPR-based functional genomic screening in neurodegeneration: mechanistic insights into AD, PD, and ALS

Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are characterized by pronounced clinical and molecular heterogeneity, as well as highly interconnected pathogenic pathways. This biological complexity has long hindered efforts to systematically define disease mechanisms and to develop effective, targeted therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) based functional genomic screening technologies have emerged as powerful tools for large-scale genetic perturbation in cellular, organoid, and in vivo models, enabling unbiased interrogation of disease relevant genetic networks and the identification of potential therapeutic targets. In this review, we summarize CRISPR knockout, CRISPR interference, CRISPR activation, and in vivo screening studies in AD, PD, and ALS, with emphasis on pathological phenotypes, experimental models, cell types, validation strategies, and evidence strength. In AD, these screens have identified regulators of amyloid-β (Aβ) production, Tau homeostasis and propagation, microglial states, neuronal aging, and stress responses. In PD, they have provided insights into α-synuclein (α-syn) homeostasis, mitochondrial quality control, lysosomal trafficking, and transplanted dopaminergic neuron survival. In ALS, they have identified modifiers of C9orf72-associated toxicity, repeat-associated non-AUG translation, TAR DNA-binding protein 43 (TDP-43) inclusion formation, and ATXN2 homeostasis. Cross-disease comparison indicates recurring involvement of proteostasis, endolysosomal function, mitochondrial regulation, and cellular stress responses, although individual screening hits show limited overlap and remain strongly influenced by experimental context. Overall, CRISPR-based screening provides a useful framework for identifying candidate disease modifiers, but further validation across complementary human-relevant and in vivo models is required before therapeutic translation.

430. Investigating cellular phenotypes and genotypes of a preclinical model relevant to schizophrenia using single-cell RNA sequencing

Abstract Background Schizophrenia is a highly heritable neurodevelopmental disorder characterised by disruptions in cortical circuitry and excitatory-inhibitory balance. Converging genetic and post-mortem evidence implicates early dysfunction of GABAergic interneurons, and transcription factor ARX plays a critical role in interneuron differentiation and migration. Mutations in ARX are associated with a spectrum of neurodevelopmental disorders, including epilepsy, autism and schizophrenia. Our laboratory identified a rare missense variant (ARX R264Q) in a female patient with schizophrenia. To investigate the developmental impact of this mutation on phenotypes relevant to schizophrenia a mouse model of the mutation that was developed using CRISPR-cas9 technology. The mouse shows behavioural and electrophysiological phenotypes relevant to schizophrenia, including disrupted sensorimotor processing and reversal learning, and social deficits coupled with altered gamma power during the aforementioned tasks. Aims & Objectives To understand how this mutation could lead to the above behavioural and electrophysiological phenotypes this study now aimed to characterise cell-type-specific transcriptional changes induced by the ArxR264Q mutation during early postnatal brain development using single-cell RNA sequencing (scRNA-seq). We sought to (i) identify transcriptionally distinct brain cell populations from WT and ArxR264Q mice, (ii) assess differential gene expression between mutant and wild-type mice within specific cell types, and (iii) define disrupted biological pathways that the identified differentially expressed genes are enriched in. We hypothesised that ArxR264Q mice would exhibit disrupted gene expression in pathways related to neuronal development, with pronounced effects in GABAergic interneuron populations. Method Whole brains from wild-type and ArxR264Q mutant mice were collected at postnatal days 3-4 and processed for multiplexed scRNA-seq using 10x Genomics technology. Data were analysed bioinformatically using Cellranger and Seurat. Cells were clustered into transcriptionally distinct populations and annotated using GPT-4o annotations. Differential gene expression was assessed within each cell type between ArxR264Q mutants and wildtypes, followed by gene set enrichment analyses to identify affected biological pathways that those differentially expressed genes act on. Results Distinct subtypes were identified in neuronal, glial and progenitor populations. Differential expression analyses revealed prominent transcriptional alterations in GABAergic interneurons, layer 4-5 excitatory neurons, developing GABAergic interneurons, and developing excitatory neurons. Enriched pathways included neurogenesis, cell migration, cell adhesion, and neuronal differentiation. Key genes implicated in interneuron migration and GABAergic function, including Cxcl12, Slc6a13, and Slc32a1, were differentially expressed in mutant mice. Additional dysregulation of developmental regulators such as Sox9, Nrp1, and Sema3a suggested broader effects on neuronal fate specification and circuit assembly. Discussion & Conclusions These findings demonstrate that the ArxR264Q mutation induces early, cell-specific transcriptional dysregulation affecting pathways critical for cortical interneuron development. This work provides mechanistic insight into how a schizophrenia-associated ARX variant may perturb early neurodevelopmental trajectories, potentially contributing to later behavioural abnormalities relevant to schizophrenia such as disrupted social behaviour, delayed reversal learning and abnormal acoustic startle, as well as disrupted neural network firing during these tasks. Future work will screen the key genes identified from this study for potentially druggable candidate targets for treating interneuronopathies commonly found in neurodevelopmental disorders.

High-throughput platforms for genetic perturbation screening using CRISPR/Cas9 in human iPSC-derived macrophages for drug discovery