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Cited by 283Pluripotent Stem Cells ResearchGenetics and Neurodevelopmental DisordersAutism Spectrum Disorder Research

Single-cell brain organoid screening identifies developmental defects in autism

Open access
Li, Chong, Fleck, Jonas Simon, Martins-Costa, Catarina, Burkard, Thomas R., Themann, Jan, Stuempflen, Marlene, Peer, Angela Maria, Vertesy, Ábel +7 more·2023·Nature
DOI 10.1038/s41586-023-06473-yPMC PMC10499611

Published version of a preprint

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Abstract

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders 1–4 . Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR–Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

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Written from the paper’s full text.

TL;DR

The authors developed CHOOSE, a pooled CRISPR–single-cell RNA sequencing system in mosaic human cerebral organoids, and used it to screen 36 high-risk autism spectrum disorder (ASD) genes for developmental loss-of-function phenotypes. They identify vulnerable progenitor and neuronal cell types, ASD-associated regulatory modules, and show that ARID1B perturbation expands ventral progenitors and shifts their fate toward early oligodendrocyte precursor cells, with validation in patient-derived organoids and prenatal MRI.

Key findings

  • Perturbation of 36 high-risk ASD genes affected cell fate: 24 perturbations significantly changed the dorsal-to-ventral cell ratio, with 21 of 24 lowering it; 23 perturbations changed abundance of at least one cell type (CMH test, FDR < 0.05).
  • IPC depletion was a convergent effect in 12 perturbations; v-RGC and/or ccv-RGC enrichment occurred in 10 perturbations; INP enrichment occurred in 10 perturbations; L2/3 excitatory neurons were depleted in the majority of perturbations.
  • Across perturbations, 2,071 differentially expressed genes were detected; top DEGs were significantly enriched in SFARI ASD risk genes (approximately twofold, Fisher exact test P < 10−5) but not in intellectual disability risk genes.
  • ARID1B perturbation enriched v-RGCs and increased their transition probability toward early OPCs (two-sided Wilcoxon P = 7.7 × 10−8); patient iPS cell-derived ventralized organoids showed increased OLIG2+, DLX2+ and DLX2+OLIG2+ cells, and prenatal MRI showed enlarged ganglionic eminence in an ARID1B-mutant patient.

Methods

  • CHOOSE combines barcoded dual sgRNA lentiviral libraries, inducible eCas9 in hES cells, and mosaic cerebral organoids; the screen targeted 36 SFARI score 1 ASD genes with verified sgRNA pairs.
  • Single-cell transcriptomics was performed on 4-month-old organoids: 49,754 cells in the full CHOOSE dataset, with 35,203 control/uninduced cells used for annotation; data came from 14 independent organoid pools, 3–7 organoids per pool, 65 organoids total, across three batches.
  • Analyses included differential gRNA representation, CMH-based differential abundance, differential expression, single-cell multiome-based GRN inference with Pando, CellRank fate mapping, immunohistochemistry, patient iPS cell-derived organoids, and fetal MRI with 3D reconstruction.

Limitations

  • The paper states the system lacks certain brain cell types such as microglia and does not include medial ganglionic eminence-derived interneurons.
  • The paper states it does not know whether perturbed cells are heterozygous or homozygous for each mutation, and that transient effects of ASD risk gene perturbations may not be captured.
  • The paper notes mutant cells remained at low percentages during development, averaging 21.8% on day 120, which could limit mutant cell–cell interactions within mosaic tissues.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-023-06473-y

Cites

105 of 105 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

31 of 42 cited sources checked · 18 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The dual gRNA located within the 3′ long terminal repeat is transcribed by RNA polymerase II and captured by single-cell RNA sequencing assays.

    “The dual gRNA is located within the 3′ long terminal repeat (LTR) and is thus transcribed by RNA polymerase II to be captured by scRNA-seq assays17.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome read-out. Nat. Methods 14, 297–301 (2017). source

      In “Direct detection of gRNAs from single-cell transcriptome data” (p. 2), the paper places an hU6-gRNA cassette in the 3′ LTR and says the gRNA becomes part of an RNA polymerase II transcript detectable by poly-A-enriched RNA-seq. The paper describes an individual gRNA in that cassette, however, not a dual-gRNA construct; that qualifier is unsupported. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The LGE PAX6+ interneuron signature resembles mouse olfactory bulb precursors that generate neurons redirected to white matter in primates.

    “Notably, we found a cluster of interneurons expressing MEIS2 and high levels of PAX6 (LGE PAX6+ interneurons), a signature that resembles mouse olfactory bulb precursors that were recently reported to generate neurons redirected to white matter in primates29.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Schmitz, M. T. et al. The development and evolution of inhibitory neurons in primate cerebrum. Nature 603, 871–877 (2022). source
  • Premisecitation

    Many autism spectrum disorder risk genes associated with transcriptional regulation and chromatin remodelling are crucial for cortical development.

    “Many ASD risk genes associated with transcriptional regulation and chromatin remodelling are crucial for cortical development21,22.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [21] Study, T. D. et al. Synaptic, transcriptional, and chromatin genes disrupted in autism. Nature 515, 209–215 (2014). source

      The Conclusions call chromatin remodeling and transcription critical pathways for typical development and link chromatin remodeling to neurogenesis and neural differentiation. The Integrating complementary data section uses mid-fetal neocortex as a developmental context for identifying ASD risk genes, but does not establish that many of the transcriptional and chromatin-remodeling risk genes are individually crucial for cortical development. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [22] Trevino, A. E. et al. Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution. Cell 184, 5053–5069.e23 (2021). source
  • Premisecitation

    Coexpression network analyses of autism spectrum disorder genes indicate that developmental defects associated with autism spectrum disorder may arise during fetal stages.

    “Moreover, coexpression network analyses of ASD genes indicate that the developmental defects associated with ASD may arise during fetal stages5,6, periods that are difficult to investigate.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [5] Willsey, A. J. et al. Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism. Cell 155, 997–1007 (2013). source
    • cites [6] Parikshak, N. N. et al. Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism. Cell 155, 1008–1021 (2013). source
  • Premisecitation

    CRISPR screening coupled with single-cell transcriptomic readout provides unprecedented resolution for phenotypic characterization.

    “Although CRISPR screening coupled with single-cell transcriptomic readout provides unprecedented resolution for phenotypic characterization14–16, such approaches have not been fully explored in organoids.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [14] Dixit, A. et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866.e17 (2016). source
    • cites [15] Jaitin, D. A. et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Cell 167, 1883–1896.e15 (2016). source
    • cites [16] Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016). source
  • May combine two claims. 38
  • Classified as probably not a substantial claim. 23
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    Organoids are limited by phenotypic variability and low throughput.

    “Although organoids have been used to investigate disease-associated genes9–11, they are limited by phenotypic variability and low throughput.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [9] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). source
    • cites [10] Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015). source
    • cites [11] Paulsen, B. et al. Autism genes converge on asynchronous development of shared neuron classes. Nature 602, 268–273 (2022). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previously established protocols reproducibly generate human telencephalon organoids.

    “We used previously established protocols that reproducibly generate human telencephalon organoids23,24 (Extended Data Fig. 5a–c).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [23] Lancaster, M. A. et al. Guided self-organization and cortical plate formation in human brain organoids. Nat. Biotechnol. 35, 659–666 (2017). source
    • cites [24] Eichmüller, O. L. et al. Amplification of human interneuron progenitors promotes brain tumors and neurological defects. Science 375, eabf5546 (2022). source

      The Organoid generation section describes generating human organoids and patterning them toward dorsal or ventral brain regions using previously described methods. The CLIP cells section also reports using patterning protocols to generate dorsal and ventral forebrain organoids. Our copy does not establish that those protocols reproducibly generate organoids, so that part of the claim remains unsupported. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Aberrant cell proliferation during brain development has been suggested to contribute to autism spectrum disorder pathology.

    “Aberrant cell proliferation during brain development has been suggested to contribute to ASD pathology33.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] Courchesne, E. et al. The ASD Living Biology: from cell proliferation to clinical phenotype. Mol. Psychiatr. 24, 88–107 (2019). source

  • Classified as probably not a substantial claim.

    Premisecitation

    CRISPR screening combined with organoids has revealed power for discovering new gene functions.

    “Recent studies combining CRISPR screening technology with organoids have revealed the power of such strategies for discovering new gene functions12,13.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [12] Esk, C. et al. A human tissue screen identifies a regulator of ER secretion as a brain-size determinant. Science 370, 935–941 (2020). source
    • cites [13] Michels, B. E. et al. Pooled in vitro and in vivo CRISPR-Cas9 screening identifies tumor suppressors in human colon organoids. Cell Stem Cell 26, 782–792.e7 (2020). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Human brain tissues exhibit generation of clones with highly variable sizes both in vivo and in vitro.

    “It is important to note that the development of human brain tissues exhibits the generation of clones with highly variable sizes both in vivo and in vitro12,19.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [12] Esk, C. et al. A human tissue screen identifies a regulator of ER secretion as a brain-size determinant. Science 370, 935–941 (2020). source
    • cites [19] Bizzotto, S. et al. Landmarks of human embryonic development inscribed in somatic mutations. Science 371, 1249–1253 (2021). source

      The indexed passage (labeled Abstract; no page supplied) reports mosaic fractions ranging from 5% to 26% across cerebral cortex samples, supporting highly variable clonal amplification in human brain tissue in vivo. Our copy does not report variable clone sizes in brain tissue grown in vitro, so that part of the claim is not supported by this source. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Brain organoids recapitulate early brain development and generate diverse cell types found in vivo.

    “Brain organoids recapitulate early brain development and generate diverse cell types found in vivo9.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    OLIG2 is known to regulate progenitor self-renewal at earlier developmental stages and is a master regulator for oligodendrocyte lineage specification in the ventral telencephalon.

    “This is an interesting finding given that OLIG2 is known to regulate progenitor self-renewal at earlier developmental stages and is a master regulator for oligodendrocyte lineage specification in the ventral telencephalon44,45.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [44] Petryniak, M. A., Potter, G. B., Rowitch, D. H. & Rubenstein, J. L. R. Dlx1 and Dlx2 control neuronal versus oligodendroglial cell fate acquisition in the developing forebrain. Neuron 55, 417–433 (2007). source
    • cites [45] Sun, Y. et al. Phosphorylation state of Olig2 regulates proliferation of neural progenitors. Neuron 69, 906–917 (2011). source

      The Introduction (page unavailable) says Olig2 sustains progenitor replication competence, is required for optimal proliferation in embryonic forebrain neurospheres, and later helps specify oligodendrocyte progenitors. The Results (page unavailable) also report that disrupting Olig2 nearly abolishes the oligodendrocyte lineage. Our copy does not establish the claim’s specific location, the ventral telencephalon, for that lineage-specification role. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CHCHD2 encodes a mitochondrial protein, and its downregulation has been observed in neurons from postmortem brains of patients with autism spectrum disorder.

    “CHCHD2 encodes a mitochondrial protein, and its downregulation has been observed in neurons from the postmortem brains of patients with ASD34.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [34] Velmeshev, D. et al. Single-cell genomics identifies cell type–specific molecular changes in autism. Science 364, 685–689 (2019). source

      The indexed text (section labeled Abstract; page unavailable) reports downregulated neuronal genes in postmortem ASD samples. It does not identify CHCHD2 among those genes or say that CHCHD2 encodes a mitochondrial protein, so it does not support the gene-specific claim. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Notable perturbation-specific gene ontology terms include ribosome assembly (SETD5), mitochondrion organization (FOXP1), lipid homoeostasis (IRF2BPL), autophagosome maturation (KAT2B), and cilium development (MECP2).

    “Some notable examples include the gene ontology terms ribosome assembly (SETD5 perturbation)36, mitochondrion organization (FOXP1 perturbation)37, lipid homoeostasis (IRF2BPL perturbation)38, autophagosome maturation (KAT2B perturbation)39 and cilium development (MECP2 perturbation)40.”
    Quote in the paperNot a substantial claim5 of 5 sources checked
    • cites [36] Nakagawa, T. et al. The autism-related protein SETD5 controls neural cell proliferation through epigenetic regulation of rDNA expression. iScience 23, 101030 (2020). source

      The Results section, “Impaired Expression of Ribosome-Related Genes in the Setd5+/– Mouse Brain” (page unavailable), reports GO enrichment of ribosomal-protein genes among genes down-regulated after SETD5 reduction. It also discusses ribosome assembly, but does not identify “ribosome assembly” as an enriched GO term. The other gene–term pairs have separate citations and are not attributed to this source. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [37] Wang, J. et al. Mitochondrial dysfunction and oxidative stress contribute to cognitive and motor impairment in FOXP1 syndrome. Proc. Natl Acad. Sci. USA 119, e2112852119 (2022). source
    • cites [38] Chen, H.-H. et al. IRF2BP2 reduces macrophage inflammation and susceptibility to atherosclerosis. Circ. Res. 117, 671–683 (2015). source

      The section “IRF2BP2-Deficient Macrophages Have an Increased Propensity for Foam Cell Formation” (page unavailable) reports altered lipid accumulation, cholesterol uptake, and cholesterol efflux after IRF2BP2 deletion. That supports a link between IRF2BP2 perturbation and lipid handling, but the cited paper does not establish the claimed lipid-homoeostasis gene ontology term for IRF2BPL perturbation; IRF2BPL is a different gene. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [39] Jia, Y.-L. et al. P300/CBP-associated factor (PCAF) inhibits the growth of hepatocellular carcinoma by promoting cell autophagy. Cell Death Dis. 7, e2400–e2400 (2016). source

      Citation 39 is attached to the KAT2B–autophagosome maturation example. The Results section reports that perturbing PCAF affects autophagy and autophagosome formation in hepatocellular carcinoma cells, but it does not identify autophagosome maturation as a perturbation-specific gene ontology term or establish an effect specifically on maturation. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [40] Frasca, A. et al. MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders. EMBO Mol. Med. 12, e10270 (2020). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    Emulsion PCR was used to reduce PCR bias and prevent chimeric PCR products.

    “Emulsion polymerase chain reaction (PCR) was used to recover gRNA and UCB sequences from plasmid libraries, genomic DNA extracted from lentivirus-infected hES cells and cells sorted from CHOOSE mosaic organoids as well as 10× single-cell complementary DNA libraries to reduce PCR bias and to prevent the generation of chimeric PCR products56,57.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [56] Omelina, E. S., Ivankin, A. V., Letiagina, A. E. & Pindyurin, A. V. Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries. BMC Genom. 20, 536 (2019). source

      The Background and Results (pages unavailable) support using optimized emulsion PCR to minimize chimeric products from MPRA plasmid libraries. The paper does not establish that it reduces PCR bias, and its results do not address the gRNA, genomic-DNA, organoid, or single-cell libraries named in the citing sentence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [57] Williams, R. et al. Amplification of complex gene libraries by emulsion PCR. Nat. Methods 3, 545–550 (2006). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ASD risk gene coexpression networks are enriched in upper-layer neurons during development and these neurons are preferentially affected in ASD patients.

    “This aligns with the observation that ASD risk gene coexpression networks are enriched in upper-layer neurons during development and that these neurons are preferentially affected in ASD patients6,34.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [6] Parikshak, N. N. et al. Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism. Cell 155, 1008–1021 (2013). source

      The Results (page unavailable) link ASD-risk coexpression modules to upper-layer neuron markers and report that genes recurrently mutated in ASD probands tend to be expressed in superficial layers. The Discussion (page unavailable) treats disruption of upper-layer neurons as a prediction, not a direct observation that those neurons are preferentially affected in patients; it also says the strongest layer enrichment was found in adult tissue. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [34] Velmeshev, D. et al. Single-cell genomics identifies cell type–specific molecular changes in autism. Science 364, 685–689 (2019). source

      The paper reports that ASD-associated genes and molecular changes are enriched in upper-layer cortical neurons, and that gene expression in layer 2/3 neurons is especially affected in ASD patients (Abstract; page not provided). It does not report an analysis showing that ASD risk-gene coexpression networks are enriched in those neurons during development; it refers more generally to previous studies of fetal cell-type convergence. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    A developmental gene regulatory network of cerebral organoids was constructed from single-cell transcriptomes and chromatin modalities, and autism spectrum disorder-associated and perturbation-enriched regulatory modules were identified.

    “We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dorsal progenitors included radial glial cells (VIM), cycling radial glial cells (ASPM), outer radial glial cells (HOPX), and intermediate progenitor cells (EOMES).

    “Among the cells with dorsal identity, we identified radial glial cells (RGCs; VIM), cycling RGCs (ASPM), outer RGCs (oRGCs; HOPX) and intermediate progenitor cells (IPCs; EOMES).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    These progenitors differentiated into excitatory neurons with specific layer identities, including L5/6 (BCL11B), L6 cortical thalamic projection neurons (FOXP2, TLE4), L4 (RORB, UNC5D, NR2F1), and L2/3 (SATB2).

    “These progenitors differentiated into excitatory neurons with specific layer identities, including layer 5/6 neurons (L5/6; BCL11B), L6 cortical thalamic projection neurons (FOXP2, TLE4)25, L4 neurons (RORB, UNC5D, NR2F1)26,27 and L2/3 neurons (SATB2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Ventral radial glial cells differentiated into interneuron precursor cells (DLX2), which generated lateral ganglionic eminence interneurons (MEIS2) or caudal ganglionic eminence interneurons (NR2F2).

    “Ventral radial glial cells (v-RGCs; cell cycling ventral RGCs, ccv-RGCs) differentiated into interneuron precursor cells (INPs; DLX2), which generated interneurons with lateral ganglionic eminence (LGE) origin (LGE-IN; MEIS2) or caudal ganglionic eminence (CGE) origin (CGE-IN; NR2F2)28.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Perturbing ARID1B expands ventral radial glia cells and increases their transition to early oligodendrocyte precursor cells, a phenotype verified in ARID1B patient-derived induced pluripotent stem cell organoids.

    “Specifically, perturbing ARID1B expands ventral radial glia cells and increases their transition to early oligodendrocyte precursor cells (OPCs), a phenotype we verify in brain organoids generated from ARID1B patient-derived induced pluripotent stem cell (iPS cell) lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using single-cell multiomic data, a developmental gene regulatory network of cerebral organoids was constructed and autism spectrum disorder-enriched regulatory hubs connected to dysregulated genes were identified.

    “Using single-cell multiomic data, we construct a developmental gene regulatory network (GRN) of cerebral organoids and identify ASD-enriched regulatory hubs connected to the genes that are dysregulated in response to genetic perturbations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype confirmed in patient-specific induced pluripotent stem cell-derived organoids.

    “Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In ventral telencephalic cells, CREB5, MEIS2, NFIA, and OLIG1 were most strongly affected, whereas dorsal telencephalon-specific DEGs were strongly enriched in SATB2, BACH2, MEF2C, and EOMES modules.

    “In the ventral telencephalic cells, CREB5, MEIS2, NFIA and OLIG1 were most strongly affected, whereas dorsal telencephalon-specific DEGs were strongly enriched in SATB2, BACH2, MEF2C and EOMES modules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gene regulatory subnetworks of OLIG1 and EOMES were presented, both enriched in autism spectrum disorder risk genes and strongly affected by autism spectrum disorder genetic perturbations.

    “We finally present gene regulatory subnetworks of OLIG1 and EOMES, which are both enriched in ASD risk genes and strongly affected by ASD genetic perturbations (Fig. 3g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Six perturbations specifically targeted one cell type without affecting others, including ADNP (L2/3 enrichment), POGZ (RGC enrichment), and SETD5 (ccv-RGC enrichment).

    “On the other hand, six perturbations specifically targeted one cell type without affecting others, including ADNP (L2/3 enrichment), POGZ (RGC enrichment) and SETD5 (ccv-RGC enrichment).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunohistochemical analyses detected significantly decreased EOMES+ IPCs in KMT2C and PHF3 perturbations and increased DLX2+ INPs in KMT2C, MED13, PHF3, and TBL1XR1 perturbations.

    “Consistent with the screen data, we detected significantly decreased EOMES+ IPCs in KMT2C and PHF3 perturbations (Extended Data Fig. 11) and increased DLX2+ INPs in KMT2C, MED13, PHF3 and TBL1XR1 perturbations (Extended Data Fig. 12).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Terminal fate probabilities revealed a distinct differentiation trajectory from ventral progenitors towards early oligodendrocyte precursor cells (OLIG2, PDGFRA) and branching of interneuron precursor cells (DLX2) into different inhibitory neuronal fates (DLX5).

    “We visualized the terminal fate probabilities for each cell as a circular projection, which revealed a distinct differentiation trajectory from ventral progenitors towards early OPCs (OLIG2, PDGFRA) and a branching of INPs (DLX2) into different inhibitory neuronal fates (DLX5) (Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Interneuron analysis revealed enrichment of both CGE-INs and LGE-INs in three perturbations and enrichment of only CGE-INs or LGE-INs in four perturbations.

    “Analysis of interneurons revealed enrichment of both CGE-INs and LGE-INs in three perturbations and enrichment of only CGE-INs or LGE-INs in four perturbations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In control versus ARID1B perturbation, v-RGCs positive for DLX2 are 68.6 versus 81.5%, for OLIG2 19.6 versus 36.2%, and for both 9.8 versus 30.6%.

    “Bar graph shows cells within the v-RGCs that are positive for DLX2 (68.6 versus 81.5%), OLIG2 (19.6 versus 36.2%) and both (9.8 versus 30.6%) in control versus ARID1B perturbation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bulk genomic DNA analysis revealed high clonal complexity in both eCas9-uninduced and eCas9-induced cells, with homogenous distribution only in uninduced cells.

    “Bulk analysis of the genomic DNA with a higher cell number input (50,000–150,000) revealed high clonal complexity in both eCas9-uninduced and eCas9-induced cells, with a homogenous distribution only in uninduced cells (Extended Data Fig. 4g,h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ARID1B-perturbed cells were strongly enriched in the oligodendrocyte precursor cell trajectory and had a higher percentage of OLIG2+ ventral radial glial cells.

    “We found that ARID1B-perturbed cells were strongly enriched in the OPC trajectory and had a higher percentage of OLIG2 + v-RGCs (Fig. 4c,d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Genes dysregulated in both dorsal and ventral populations, as well as those specifically dysregulated in one population, were identified.

    “Additionally, we could identify genes dysregulated in both dorsal and ventral populations, as well as those specifically dysregulated in one population (KMT2C, LEO1 perturbation) (Extended Data Fig. 13a).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Prenatal MRI of patient 2 at GW22 and GW31 revealed an enlarged ganglionic eminence compared with multiple age-matched controls after normalization to cortical and total brain volume.

    “To further explore the potential consequences of such defects in patients, we analysed the prenatal brain structure of patient 2 at two gestation stages (gestational weeks (GW) 22 and GW31) by intrauterine super-resolution magnetic resonance imaging (MRI).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transition probabilities of control (n = 51) and ARID1B-perturbed (n = 46) ventral progenitor cells towards OPCs differ, with two-sided Wilcoxon P = 7.7 × 10−8.

    “Box plots showing transition probabilities of control (n = 51) and ARID1B-perturbed (n = 46) ventral progenitor cells towards OPCs. The centre lines represent the medians, the box limits show the 25–75% interquartile ranges and the whiskers indicate 1.5× the interquartile ranges. Two-sided Wilcoxon test P value = 7.7 × 10−8.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A telencephalon developmental trajectories-based GRN was constructed and ASD-associated regulatory modules were identified in dorsal and ventral cell populations.

    “Furthermore, we constructed a telencephalon developmental trajectories-based GRN and identified ASD-associated regulatory modules in dorsal and ventral cell populations.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Enhanced specificity SpCas9 (eCas9) has substantially reduced off-target effects.

    “we used a human embryonic stem cell (hES cell) line expressing an enhanced specificity SpCas9 (eCas9), which has substantially reduced off-target effects and is controlled by an upstream loxp stop element12”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Esk, C. et al. A human tissue screen identifies a regulator of ER secretion as a brain-size determinant. Science 370, 935–941 (2020). source

  • May combine two claims.

    Study design and conductown data

    An average of 2,770 unique clones per perturbation were obtained and used to generate mosaic embryoid bodies.

    “Using this strategy, we obtained an average of 2,770 unique clones for each perturbation, which we used to generate mosaic embryoid bodies (Fig. 1e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    gRNAs were homogeneously distributed in the plasmid library and human embryonic stem cells, and this was maintained after embryoid body formation.

    “Our analysis indicated homogeneous distribution of the gRNAs in both the plasmid library and hES cells, which was maintained after the formation of embryoid bodies (Fig. 1d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Many perturbation-specific gene ontology terms confirm previous studies, supporting the power of detecting complex biological phenotypes with the CHOOSE system.

    “We also observed many perturbation-specific gene ontology terms, many of which confirm previous studies, further supporting the power of detecting complex biological phenotypes with the CHOOSE system (Extended Data Fig. 13b and Supplementary Data 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Leveraging gene regulatory network inference from multiomic data, autism spectrum disorder-associated transcription factor modules during cortical development and critical regulatory hubs underlying detected gene expression changes were identified.

    “Leveraging GRN inference from multiomic data, we further identified ASD-associated TF modules during cortical development and critical regulatory hubs underlying the detected gene expression changes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Clone complexity analysis recovered on average 125 clones per perturbation, distributed across all libraries.

    “On average, we recovered 125 clones for each perturbation and found that the clones were distributed across all libraries (Extended Data Fig. 4c,d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The study characterized gene expression changes for each genetic perturbation in both dorsal and ventral telencephalon and uncovered molecular programs shared between different perturbations.

    “Thus, we have characterized gene expression changes for each genetic perturbation in both dorsal and ventral telencephalon and uncovered molecular programs shared between different perturbations.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Age-matched control subjects were included only if they presented an absence of confounding comorbidities, including structural cerebral or cardiac anomalies or fetal growth restriction.

    “Age-matched control subjects were identified and included if they presented an absence of confounding comorbidities, including structural cerebral or cardiac anomalies or fetal growth restriction.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The superresolution data were quality assessed, and only cases that met high-quality standards (score of less than or equal to two of five) were included in the analysis.

    “The resulting superresolution data were quality assessed, and only cases that met high-quality standards (score of less than or equal to two of five) were included in the analysis.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    A background distribution of enrichment effects was computed from randomly permuted gRNA labels.

    “We additionally computed a background distribution of enrichment effects from randomly permuted gRNA labels.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No statistical methods were used to predetermine sample size unless specified.

    “No statistical methods were used to predetermine sample size unless specified.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    The CHOOSE system provides a robust, precisely controlled screening strategy.

    “As the CHOOSE system provides a robust, precisely controlled screening strategy, we anticipate that it will be widely applied beyond brain organoids to study disease-associated genes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductnone given

    The system lacks certain brain cell types, such as microglia, and does not include interneurons derived from the medial ganglionic eminence.

    “First, our system lacks certain brain cell types, such as microglia, and does not include interneurons derived from the medial ganglionic eminence.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Differential expression analysis treated library, cell_type and n_UMI as covariates in the model.

    “Tests were performed on log-normalized transcript counts Y while treating library, cell_type and n_UMI as covariates in the model:Yi≈n_UMI+library+cell_type+condition.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    High-quality cells were filtered based on number of genes detected (minimum 1,000, maximum 8,000), removing cells with high mitochondrial (less than 15%) or ribosomal (less than 20%) mRNA content.

    “Next, we filtered high-quality cells based on the number of genes detected (minimum 1,000, maximum 8,000), removing cells with high mitochondrial (less than 15%) or ribosomal (less than 20%) messenger RNA content.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The isogenic control cell line for patient 1 was verified by PCR and Sanger sequencing to determine efficient repair of the mutation.

    “After colony expansion, gDNA was extracted using DNA QuickExtract Solution (Lucigen), followed by PCR and Sanger sequencing to determine efficient repair of the mutation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No blinding and randomization were used unless specified.

    “No blinding and randomization were used unless specified.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Differential abundance testing used a CMH test stratified by library to control for confounding effects through differential gRNA sampling in different libraries.

    “To control for confounding effects through differential gRNA sampling in different libraries, we used a CMH test stratified by library.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Samples with more than 85% viability were processed.

    “Samples with more than 85% viability were processed.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Fourteen scRNA-seq libraries obtained from independent pools of organoids were used as replicates from three batches.

    “For this, we used 14 scRNA-seq libraries obtained from independent pools of organoids as replicates from three batches.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Infection rate was controlled to be lower than 5% to prevent multiple infections.

    “Infection rate was controlled to be lower than 5% to prevent multiple infections18”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Doench, J. G. Am I ready for CRISPR? A user’s guide to genetic screens. Nat. Rev. Genet. 19, 67–80 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All cell lines were authenticated using a short tandem repeat assay, tested for genomic integrity using SNP array genotyping, and routinely tested negative for mycoplasma.

    “All cell lines were authenticated using a short tandem repeat assay, tested for genomic integrity using single-nucleotide polymorphism (SNP) array genotyping and routinely tested negative for mycoplasma.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The enrichment of v-RGCs and ccv-RGCs, the higher transition probability of v-RGCs to early OPCs, and the increased proportion of OLIG2+ cells in the screen and in organoids from two patient iPS cell lines suggest that ARID1B perturbation leads to abnormal ventral progenitor expansion and aberrant cell fate specification.

    “Taken together, the enrichment of v-RGCs and ccv-RGCs (Fig. 2a), the higher transition probability of v-RGCs to early OPCs, and the increased proportion of OLIG2 + cells in our screen and in organoids generated from two patient iPS cell lines all suggest that ARID1B perturbation leads to abnormal ventral progenitor expansion and aberrant cell fate specification (Fig. 4i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Both convergent and divergent mechanisms contribute to ASD pathophysiology.

    “suggesting that both convergent and divergent mechanisms contribute to ASD pathophysiology.”
    Quote in the paperSubstantial claim
55 of 55

  • The paper relies on this without support.

    CRISPR screens in organoids are limited by low-content readouts, often using guide RNA counts to assess proliferation phenotypes.

    “However, such screens are limited by low-content readouts, which often use guide RNA (gRNA) counts to assess proliferation phenotypes when challenged with genetic perturbations.”

  • The paper relies on this without support.

    CRISPR screening coupled with single-cell transcriptomic readout has not been fully explored in organoids.

    “Although CRISPR screening coupled with single-cell transcriptomic readout provides unprecedented resolution for phenotypic characterization14–16, such approaches have not been fully explored in organoids.”

  • The paper relies on this without support.

    The feasibility of single-cell perturbation screening in heterogeneous tissues that undergo long-term differentiation and consist of diverse cell types remains unclear.

    “The feasibility of single-cell perturbation screening in heterogeneous tissues that undergo long-term differentiation and consist of diverse cell types remains unclear.”

  • The paper relies on this without support.

    Successful genome editing causes frameshift mutations, resulting in loss of blue fluorescent protein fluorescence, enabling quantitative evaluation of guide RNA efficiency.

    “Successful genome editing causes frameshift mutations, resulting in the loss of blue fluorescent protein (BFP) fluorescence, which enables the quantitative evaluation of gRNA efficiency”

  • The paper relies on this without support.

    OLIG1 is preferentially expressed in the ventral telencephalon and is a key regulator for interneuron and oligodendrocyte lineages.

    “Oligodendrocyte transcription factor 1 (OLIG1) is preferentially expressed in the ventral telencephalon and is a key regulator for interneuron and oligodendrocyte lineages.”

  • The paper relies on this without support.

    EOMES is a key transcription factor for the fate specification of intermediate progenitor cells in the dorsal telencephalon.

    “Eomesodermin (EOMES) is a key TF for the fate specification of IPCs in the dorsal telencephalon.”
6 of 6

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • ARID1B control mutation has two identifiers

    Minor

    The Fig. 4f caption identifies the repaired control as c.220dupG, whereas the patient mutation and the control identified in the quantification methods are c.2201dupG.

  • Ventral progenitor enrichment cites the gRNA-representation panel

    Minor

    The text cites Fig. 2a for enrichment of v-RGCs and ccv-RGCs, but the figure caption says panel a shows gRNA representation. The caption assigns cell-type enrichment to panel c.

2 of 2

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Authors
Li, C., Fleck, J. S., Martins-Costa, C., Burkard, T. R., Themann, J., Stuempflen, M., Peer, A. M., Vertesy, Á., Littleboy, J. B., Esk, C., Elling, U., Kasprian, G., Corsini, N. S., Treutlein, B., & Knoblich, J. A.
Affiliations
  • Institute of Molecular Biotechnology (AT)
  • ETH Zurich (CH)
  • Medical University of Vienna (AT)
Corresponding author
  • Li, Chong
  • Treutlein, Barbara
  • Knoblich, Juergen A.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2023-09-13
Volume
621
Issue
7978
Pages
373-380
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-023-06473-y
PMC
PMC10499611
OpenAlex
W4386697296
Semantic Scholar
7794beca1a3f20e084766fc84bcb357f90f7f608
Cited by
283
References
104
Citations by year
2026: 80 · 2025: 108 · 2024: 74 · 2023: 18 · 2022: 3
FWCI
20.75
Type
journal article
Language
en
Update notices
correction (2023-11-14)
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • National Science Foundation
  • Silicon Valley Community Foundation (CZF2019-002440)
  • Simons Foundation Autism Research Initiative (724430)
  • Boehringer Ingelheim Fonds
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (192604, 310030)
  • Österreichischen Akademie der Wissenschaften (SFB F78)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
Keywords
autism spectrum disorder, brain organoids, single-cell transcriptomics, cell fate determination, gene regulatory networks, ARID1B, oligodendrocyte precursor cells, excitatory neurons, neurodevelopmental disorders, susceptibility genes
MeSH terms
Autism Spectrum Disorder, Gene Editing, Loss of Function Mutation, CRISPR-Associated Protein 9, Single-Cell Gene Expression Analysis, RNA, Guide, CRISPR-Cas Systems, Autistic Disorder, Brain, Developmental Disabilities, Chromatin, DNA Helicases, DNA-Binding Proteins, Humans, Mosaicism, Neurons, Nuclear Proteins, Organoids, Transcription Factors, Transcription, Genetic, Cell Lineage, CRISPR-Cas Systems

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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