Single-cell brain organoid screening identifies developmental defects in autism

Abstract

Development of the human brain involves processes that are not seen in many other species, but can contribute to neurodevelopmental disorders (1–4). Cerebral organoids can be used to investigate neurodevelopmental disorders in a human context but are limited by variability and low throughput. We have developed the CRISPR-human organoids-scRNA-seq (CHOOSE) system that utilizes verified pairs of gRNAs, inducible CRISPR/Cas9-based genetic disruption, and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Genetic perturbations of 36 high-risk autism spectrum disorder (ASD) genes related to transcriptional regulation allowed us to identify their effects on cell fate determination and discover developmental stages susceptible to ASD gene perturbations. We construct a developmental gene regulatory network (GRN) of cerebral organoids from single-cell multiomic data including transcriptome and chromatin modalities and identify ASD-associated and perturbation-enriched regulatory modules. We show that perturbing members of the BAF chromatin remodeling complex leads to an expanded population of ventral telencephalon progenitors. Specifically, the BAF subunit ARID1B controls the fate transitions of progenitors to oligodendrocyte precursor cells and interneurons, which we confirmed in patient-specific induced pluripotent stem cell (iPSC) derived organoids. Our study paves the way for phenotypically characterizing disease susceptibility genes in human organoid models with cell type, developmental trajectory, and gene regulatory network readouts.

Inferring and perturbing cell fate regulomes in human brain organoids

“…We have highlighted interesting aspects of the network, such as TF modules involved in the transition from pluripotency through neuroectoderm to a neuroepithelium, as well as the subnetworks associated with regionalized brain states. Such network analysis can guide future experiments designed to program specific neuronal states, and can be used to interpret gene perturbations in human organoids 45 . Note that current limitations include the lack of comprehensive active and repressive histone modification and chromatin conformation status across organoid development, as well as incomplete TF motif databases.…”

Understanding neural development and diseases using CRISPR screens in human pluripotent stem cell-derived cultures

“…In 2022, two separate CRISPR screens in hPSC-derived 3D brain organoid models have identified several convergent mechanisms and cellular abnormalities across different ASD-risk genes. A single-cell CRISPR screen in human telencephalon organoids examined the pooled knockout effect of 36 high-risk ASD genes involved in transcriptional control ( Li et al, 2022 ). To establish this approach, the authors transduced human ESCs carrying an inducible Cas9 cassette with a pooled sgRNA lentiviral library.…”

“…However, genome-wide screens can be costly and labor-intensive, in particular, when applied in complex models (e.g., brain organoids) or with high-content readouts (such as scRNA sequencing). Alternatively, more focused libraries can be utilized to target the druggable genome ( Tian et al, 2019 ), transcription factors ( Black et al, 2020 ), or disease-linked genes ( Esk et al, 2020 ; Li et al, 2022 ; Meng et al, 2022 ).…”

“… Recent applications of CRISPR screens using hPSC-derived neural cultures. hPSCs are transduced with a scalable pooled lentiviral sgRNA CRISPR library, and differentiated into 2D neural cultures (neural progenitor cells ( Li et al, 2019 ), neurons ( Tian et al, 2019 ; Tian et al, 2021 ), microglia ( Dräger et al, 2022 ), or astrocytes ( Leng et al, 2022 )), or 3D brain organoids/assembloids ( Esk et al, 2020 ; Fleck et al, 2022 ; Li et al, 2022 ; Meng et al, 2022 ). Screening neural cultures carrying different pooled CRISPR perturbations has revealed novel factors that regulate neurodevelopmental cell fates ( Black et al, 2020 ; Fleck et al, 2022 ), essential genes ( Tian et al, 2019 ; Tian et al, 2021 ), modifiers of cell survival under different challenges ( Li et al, 2019 ; Tian et al, 2021 ; Leng et al, 2022 ), and the cellular and molecular mechanisms that underlie neurodevelopmental and degenerative disorders ( Esk et al, 2020 ; Guo et al, 2022 ; Li et al, 2022 ; Meng et al, 2022 ).…”

Convergent impact of schizophrenia risk genes

“…Such genetic studies were typically conducted in a one-gene-at-a-time manner requiring a substantial time and resource investment, but state-of-the-art CRISPR screens now incorporate a pooled multiplexed design, minimizing experimental variability, increasing the power of the associated analyses, and more closely recapitulating the observed polygenic architecture of disease risk. Recent studies queried an overlapping set of highly penetrant loss-of-function ASD genes in vitro in human neural progenitor cells (27 genes) 24 and human brain organoids (3 and 36 genes) 25,26 , and in vivo in fetal mouse brains (35 genes) 27 and Xenopus tropicalis (10 genes) 28 , reporting convergence impacting neurogenesis 24,25,27,28 , WNT signaling 24 , and gene expression 25,27 . This approach has not yet been applied to SCZ genes, loss-of-function 29 or otherwise.…”