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Cited by 168CRISPR and Genetic EngineeringRNA regulation and diseaseVirus-based gene therapy research

CRISPR-based functional genomics for neurological disease

Open access
Kampmann, Martin·2020·Nature Reviews Neurology
DOI 10.1038/s41582-020-0373-zPMC PMC7484261
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Abstract

Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, as many lack disease-modifying treatments. A major reason for the absence of effective therapies is our limited understanding of the causative molecular and cellular mechanisms. Genome-wide association studies are providing a growing catalogue of disease-associated genetic variants, and the next challenge is to elucidate how these variants cause disease and to translate this understanding into therapies. This Review describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases. The bacterial CRISPR system can be used in experimental disease models to edit genomes and to control gene expression levels through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These genetic perturbations can be implemented in massively parallel genetic screens to evaluate the functional consequences for human cells. CRISPR screens are particularly powerful in combination with induced pluripotent stem cell technology, which enables the derivation of differentiated cell types, such as neurons and glia, and brain organoids from cells obtained from patients. Modelling of disease-associated changes in gene expression via CRISPRi and CRISPRa can pinpoint causal changes. In addition, genetic modifier screens can be used to elucidate disease mechanisms and causal determinants of cell type-selective vulnerability and to identify therapeutic targets.

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Crossref · 10.1038/s41582-020-0373-z

Cites

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Claims the paper makes

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  • Findingnone given

    The mechanisms underlying APOE ε4 interactions with sex and ethnicity are not understood.

    “The underlying mechanisms are not understood.”
    Quote in the paperNot a substantial claim
  • Findingcitation

    An arrayed longitudinal imaging screen revealed effects of gene knockdown on neuron-specific morphology, such as neurite length and branching, which could not be assessed in other cell types.

    “Third, an arrayed longitudinal imaging screens uncovered consequences of gene knockdown for neuron-specific morphological features, such as neurite length and branching patterns76, which could not have been evaluated in other cell types.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

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  • Findingcitation

    The paper reports the first large-scale CRISPR screens performed in human iPSC-derived neurons.

    “Recently, we implemented large-scale CRISPR-based genetic screens for the first time in a human iPSC-derived differentiated cell type, namely neurons76.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

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  • Findingcitation

    Housekeeping genes needed for survival in both iPSCs and neurons may have different functions in the two cell types, as their knockdown produced very different transcriptomic effects.

    “Second, housekeeping genes that were essential for survival of both iPSCs and neurons may play different roles in the two cell types, based on vastly different transcriptomic consequences of their knockdown76.”
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    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

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  • Findingcitation

    The screen identified MAP3K12/DLK, a known regulator of neuronal death.

    “CRISPR screening recovered a previously known key regulator of neuronal cell death, MAP3K12/DLK76.”
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    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

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    Findingcitation

    UBA1 was a notable example of a housekeeping gene with different transcriptomic effects in iPSCs and neurons.

    “A prominent example was the ubiquitin-activating E1 enzyme UBA176.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

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    Premisecitation

    Mouse and human microglia differ in expression of disease-relevant genes and adopt distinctly different states in human diseased brains compared with mouse models.

    “mouse and human microglia, which have recently emerged as key players in Alzheimer’s Disease and other neurodegenerative diseases, show differences in particular in the expression of disease-relevant genes19, and they adopt distinctly different states in human diseased brains compared to mouse models20.”
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    • cites [19] Gosselin, D. et al. An environment-dependent transcriptional network specifies human microglia identity. Science 356, eaal3222 (2017). source

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    • cites [20] Friedman, B. A. et al. Diverse brain myeloid expression profiles reveal distinct microglial activation states and aspects of Alzheimer’s disease not evident in mouse models. Cell Rep. 22, 832–847 (2018). source

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    Interpretationnone given

    Answers to key mechanistic questions have implications for understanding disease mechanisms and should clarify which therapeutic strategies are likely to be effective and disease-modifying.

    “Answers to these questions have important implications for our understanding of disease mechanisms, and should clarify which therapeutic strategies are likely to be effective and disease-modifying.”
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    Premisecitation

    RNA toxicity and dipeptide repeat toxicity mechanisms may synergize in C9orf72 disease.

    “Intriguingly, these two mechanisms may synergize in disease7.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Boivin, M. et al. Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders. EMBO J. 39, e100574 (2020). source

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    Interpretationnone given

    If processes driving age-associated neurological diseases are initiated early in life, therapeutic intervention may need to start similarly early.

    “if processes driving age-associated neurological diseases are initiated early in life, therapeutic intervention may need to start similarly early.”
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    Interpretationreasoning

    A major challenge is to determine which pleiotropic effects of genetic changes are relevant for disease.

    “therefore genetic changes can have pleiotropic effects, and a major challenge is to understand which of these effects are relevant for disease.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: For many human genes, the molecular and cellular functions of the gene product are not well understood, and many genes have multiple functions, so genetic changes can have pleiotropic effects.

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    Premisecitation

    Non-coding risk variants in humans often occur in non-conserved sequences, precluding direct mouse modeling, and it is challenging to determine which coding gene or genes they affect.

    “Non-coding risk variants in humans often occur in non-conserved sequences in the genome, precluding direct mouse modeling of these variants, and it is challenging to determine through which coding gene or genes these variants affect disease risk3.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Pimenova, A. A., Raj, T. & Goate, A. M. Untangling genetic risk for Alzheimer’s disease. Biol. Psychiatry 83, 300–310 (2018). source

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    Interpretationreasoning

    Understanding factors that make some neurons vulnerable and others resilient could inform therapeutic strategies aimed at increasing resilience of vulnerable neuronal populations.

    “An understanding of the factors that make some neurons vulnerable and others comparatively resilient to disease could inform therapeutic strategies aimed at increasing resilience of vulnerable neuronal populations.”
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    • Rests on an assumption: Selective vulnerability of specific cell types is a hallmark of many neurological diseases and drives disease-specific symptoms.

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    Premisecitation

    Coding variants that cause disease in humans may not cause disease in mice; the A53T alpha-synuclein mutation is linked to Parkinson's disease in humans but corresponds to the wild-type residue in mice, so mouse models rely on overexpression of human proteins.

    “Coding variants that are disease-causing in humans may not cause disease in mice: the A53T mutation in alpha-synuclein is linked to Parkinson’s Disease in humans but corresponds to the wild-type residue in mice, and mouse models of A53T alpha-synuclein rely on overexpression of human proteins21.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Giasson, B. I. et al. Neuronal α-synucleinopathy with severe movement disorder in mice expressing A53T human α-synuclein. Neuron 34, 521–533 (2002). source

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    Premisecitation

    Human dopaminergic neurons contain substantially higher dopamine levels than mouse dopaminergic neurons, and some cellular disease processes have been proposed to depend on high dopamine found only in human dopaminergic neurons.

    “dopaminergic neurons, which are selectively vulnerable in Parkinson’s Disease, contain substantially higher levels of dopamine in humans compared to mice, and some cellular disease processes have been proposed to depend on the high levels of dopamine found only in human dopaminergic neurons18.”
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    • cites [18] Burbulla, L. F. et al. Dopamine oxidation mediates mitochondrial and lysosomal dysfunction in Parkinson’s disease. Science 357, 1255–1261 (2017). source

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    Premisenone given

    Imaging technologies enable longitudinal tracking of disease progression in patient brains, but interventions to establish causality are only possible in clinical trials.

    “advances in imaging technologies, including magnetic resonance imaging and positron emission tomography with specific molecular tracers, have enabled longitudinal tracking of disease progression in patient brains, but interventions to establish causality are only possible in the context of clinical trials.”
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    Premisecitation

    Experimental results support RNA toxicity, dipeptide repeat toxicity, and C9orf72 haploinsufficiency as causes of disease in C9orf72 repeat expansion ALS and frontotemporal dementia.

    “Experimental results support toxicity of RNAs containing the expanded repeat, as well as toxicity of dipeptide repeat translated from these RNAs, but also to haploinsufficiency of the C9orf72 protein as a cause of disease6.”
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    • cites [6] Balendra, R. & Isaacs, A. M. C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat. Rev. Neurol. 14, 544–558 (2018). source

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    Interpretationreasoning

    A functional and mechanistic understanding is essential for translating human genetics findings into effective therapies.

    “A functional and mechanistic understanding will be key to translating findings from human genetics to effective therapies.”
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    Premisecitation

    Disease-associated mutations often occur in broadly expressed proteins, yet specific subpopulations are selectively affected by downstream processes, as with tau and Von Economo neurons in frontotemporal dementia.

    “Disease-associated mutations are often found in proteins that are expressed broadly (such as tau, which is expressed in all neurons), yet specific subpopulations are selectively affected by downstream processes (such as the Von Economo neurons in frontotemporal dementia10).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Seeley, W. W. et al. Early frontotemporal dementia targets neurons unique to apes and humans. Ann. Neurol. 60, 660–667 (2006). source

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    Premisecitation

    iPSCs have been derived from post-mortem human brain tissue, specifically dura mater fibroblasts.

    “More recently, iPSCs were successfully obtain from post-mortem human brain tissue, specifically dura mater fibroblasts91.”
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    • cites [91] Ridge, P. G. et al. Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer’s disease resilience. Genome Med. 9, 100 (2017). source

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    Premisecitation

    Such screens yield rich information about processes including differentiation, immune responses, stress responses and compensatory responses, and allow high-throughput mapping of regulatory elements.

    “Such screens provide rich information on cellular processes such as differentiation72, immune responses72,73,75, stress responses74 and compensatory responses76 and enable high-throughput mapping of regulatory elements77.”
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    • cites [72] Rauch, J. N. et al. LRP1 is a master regulator of tau uptake and spread. Nature 580, 381–385 (2020). source

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    • cites [74] Rousseaux, M. W. C. et al. A druggable genome screen identifies modifiers of α-synuclein levels via a tiered cross-species validation approach. J. Neurosci. 38, 9286–9301 (2018). source

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    • cites [77] Cheng, W. et al. CRISPR-Cas9 screens identify the RNA helicase DDX3X as a repressor of C9ORF72 (GGGGCC)n repeat-associated non-AUG translation. Neuron 104, 885–898 (2019). source

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    Premisereasoning

    CRISPR enables rapid generation of a site-specific nuclease by combining Cas9 or functionally similar proteins with a single guide RNA that directs it to the desired target site.

    “CRISPR enables the rapid generation of a site-specific nuclease by combining the Cas9 protein (or functionally similar proteins from other microbial species) with a single guide RNA (sgRNA) that directs Cas9 to the desired target site.”
    Quote in the paperSubstantial claim

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    Premisecitation

    Genome editing in nonhuman primates has been used to generate models of Duchenne muscular dystrophy by dystrophin disruption, microcephaly by MCPH1 disruption, and Rett syndrome by MECP2 disruption.

    “used to generate models of neurological and related diseases: disruption of the dystrophin gene to model Duchenne muscular dystrophy29, disruption of the MCPH1 gene to model microcephaly30, and disruption of the MECP2 gene to model Rett syndrome31.”
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    • cites [29] Chen, Y. et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum. Mol. Genet. 24, 3764–3774 (2015). source

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    • cites [30] Ke, Q. et al. TALEN-based generation of a cynomolgus monkey disease model for human microcephaly. Cell Res. 26, 1048–1061 (2016). source

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    • cites [31] Chen, Y. et al. Modeling Rett syndrome using TALEN-edited MECP2 mutant cynomolgus monkeys. Cell 169, 945–955 (2017). source

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    Interpretationnone given

    Experiments in animals, especially nonhuman primates, are time-consuming, costly, and ethically challenging, limiting scalability for investigating many disease-associated variants.

    “However, experiments in animals, in particular non-human primates, are time-consuming, costly, and have important ethical considerations, limiting their scalability for the investigation of the large number of disease-associated variants that are being uncovered.”
    Quote in the paperNot a substantial claim

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    Premisecitation

    Mammalian genomes can be edited by homologous recombination when a double-strand break is introduced and a DNA template with high sequence similarity is provided; an artificial template can incorporate desired DNA sequences.

    “When a double-stranded break is introduced into genomic DNA, cells can repair the break by using a DNA molecule with high sequence similarity as a template. If an artificial template is provided while the DNA break is introduced, this pathway provides a mean to incorporate desired DNA sequences into the genome34,35”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). source

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    • cites [35] Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). Together with Cong et al. (2013), this study demonstrates the use of CRISPR–Cas9-based genome engineering in human cells. source

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

    Premisecitation

    CRISPR technology is based on a prokaryotic antiviral defense system with CRISPR DNA elements and Cas proteins; CRISPRs derive from bacteriophage genomes and generate RNAs that function with Cas proteins to cleave bacteriophage genomes upon infection.

    “CRISPR technology is based on a prokaryotic antiviral defense system characterized by DNA elements termed CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins. CRISPRs are derived from bacteriophage genomes and generate RNAs that function with Cas proteins to cleave bacteriophage genomes upon infection32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009). source

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    Premisenone given

    Segmenting microscopy images of cells with complex morphologies, such as neurons and glia, is difficult, and this segmentation is needed to assign all parts of a cell to the correct sgRNA.

    “in practice, however, it is difficult to segment microscopic images of cells with complex morphologies, such as neurons and glia, which is a prerequisite for assigning all parts of a cell to the correct sgRNA.”
    Quote in the paperSubstantial claim

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    Premisecitation

    Fibroblasts can be directly reprogrammed into differentiated cells such as neurons.

    “Fibroblasts can also be directly reprogrammed into differentiated cell types such as neurons92.”
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    • cites [92] Finch, N. et al. TMEM106B regulates progranulin levels and the penetrance of FTLD in GRN mutation carriers. Neurology 76, 467–474 (2011). source

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    Interpretationreasoning

    Cancer cell line screens have limitations for neurological disease research, including that most neurological diseases involve specific cell types, that disease-relevant genes may not be expressed in the line, and that survival control differs between cancer and untransformed cells.

    “Even though such screens have been able to uncover genes that were then validated in relevant cell types such as neurons63,65,68, they have obvious limitations for the study of neurological disease.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [63] Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014). source

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    • cites [65] Hanna, R. E. et al. Massively parallel assessment of human variants with base editor screens. Preprint at bioRxiv https://doi.org/10.1101/2020.05.17.100818 (2020). source

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    • cites [68] Kramer, N. J. et al. CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat. Genet. 50, 603–612 (2018). source

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    • Rests on an assumption: Most neurological diseases involve functional changes in specific nervous system cell types, and these functions can only be properly studied in those cell types.

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    Premisecitation

    CRISPR platforms have produced much larger genetic interaction maps than RNA interference, including CRISPRa gain-of-function maps and maps combining gain- and loss-of-function perturbations.

    “This approach was pioneered in mammalian cells using RNA interference80,81, but CRISPR-based platforms have enabled the generation of substantially larger genetic interaction maps82, including CRISPRa-based gain-of-function maps83 and maps that combine gain- and loss-of-function perturbations to map directional pathways84.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [82] Tian, R. et al. CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons. Neuron 104, 239–255 (2019). This paper describes the use of a platform for large-scale CRISPR screens in neurons derived from human iPSCs. source

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    • cites [83] Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390.e19 (2019). source

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    • cites [84] Mimitou, E. P. et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat. Methods 16, 409–412 (2019). source

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    Premisecitation

    A recent neurological disease-related genetic interaction screen found FIS1 as the strongest synthetic lethal gene with C9orf72 loss, and follow-up work implicated it in inflammatory signaling.

    “A recent example for a genetic interaction screen with relevance to neurological disease is a screen for genes loss of which was synthetic lethal with C9orf72 loss88. The strongest synthetic lethal gene was FIS1, which encodes a protein that mediating mitochondrial fission and mitophagy, and follow-up experiments also pointed to a role in regulating inflammatory signaling88.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] Kampmann, M., Bassik, M. C. & Weissman, J. S. Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells. Proc. Natl Acad. Sci. USA 110, E2317–E2326 (2013). source

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

    Premisecitation

    UBA1 is broadly expressed in the human body, and its loss-of-function mutations are associated with neurodegenerative diseases.

    “Intriguingly, UBA1 is widely expressed in the human body, but loss-of-function mutations are linked to neurodegenerative diseases110.”
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    • cites [110] Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). source

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  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Motor neurons are selectively vulnerable in ALS.

    “For example, motor neurons are selectively vulnerable in amyotrophic lateral sclerosis (ALS).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Many neurological disorders, especially neurodegenerative diseases, are characterized by selective vulnerability of specific cell types.

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

    Premisecitation

    iPSC-derived cells from different lineages can be combined into defined three-dimensional cultures called assembloids.

    “Furthermore, iPSC-derived cell types from different lineages can be combined into defined three-dimensional cultures, termed assembloids9,94–98.”
    Quote in the paperNot a substantial claim0 of 6 sources checked
    • cites [9] Lin, Y. T. et al. APOE4 causes widespread molecular and cellular alterations associated with Alzheimer’s disease phenotypes in human iPSC-derived brain cell types. Neuron 98, 1141–1154 (2018). source

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    • cites [94] Chai, N. et al. Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72. Brain Res. 1728, 146601 (2020). source

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    • cites [95] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). source

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    • cites [96] Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007). source

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    • cites [97] Sproul, A. A. et al. Generation of iPSC lines from archived non-cryoprotected biobanked dura mater. Acta Neuropathol. Commun. 2, 4 (2014). source

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    • cites [98] Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010). source

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  • May combine two claims.

    Interpretationreasoning

    Determining which genes mediate a disease-associated eQTL's effect is not trivial, and even when affected genes are known, it is unclear which are disease-relevant.

    “Therefore, it is not trivial to determine the genes through which a disease-associated eQTL affects disease processes. Even if the set of genes affected in their expression by a genetic variant is known, it is not clear which of these candidate genes is relevant for disease.”
    Quote in the paperSubstantial claim

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    Interpretationreasoning

    Disease-associated cell states involve expression changes in many genes, but it is generally unclear which changes are beneficial or detrimental.

    “These disease-associated states are characterized by expression changes in numerous genes, but it is generally not clear which of these changes have either beneficial or detrimental effects in disease.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Linkage disequilibrium can hinder causal assignment to a specific genetic variant from natural human variation, and isogenic lines can overcome this.

    “Importantly, linkage disequilibrium can make it difficult to assign causality to a specific genetic variant based on natural genetic variation in the human population, and the use of isogenic lines can overcome this problem.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    CRISPR-induced DNA breaks are especially toxic to iPSCs, lowering survival and potentially confounding observed phenotypes.

    “However, CRISPR-induced DNA breaks are particularly toxic to iPSCs111, reducing cell survival and potentially confounding the observed phenotypes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [111] Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Most differentially expressed factors likely reflect neuronal functional specializations, and it is unclear which are causal for vulnerability to specific diseases.

    “However, most differentially expressed factors are likely to reflect the different functional specializations of neurons, and it is not clear which of these factors are causal determinants of vulnerability to specific diseases.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    ALS risk genes are not expressed only in motor neurons, suggesting that factors in the motor neuron environment increase their vulnerability.

    “Intriguingly, the expression of ALS risk genes is not restricted to motor neurons, indicating that specific factors in the cellular environment of motor neurons make them more vulnerable to ALS.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premisenone given

    eQTLs can affect nearby and distant genes, and their effects can be cell-type and cell-state specific.

    “eQTLs can affect the expression of nearby, but also distant genes, and their effect can be cell-type and cell-state specific.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Genetic screens can be attractive alternatives to pharmacological screening because some phenotypes can be run as pooled screens, scalable to genome-wide without automation.

    “First, while small-molecule screens need to be implemented in an arrayed format and therefore require automation infrastructure, genetic screens for some phenotypes can be implemented as pooled screens, making them scalable to genome-wide screens without requiring automation.”
    Quote in the paperSubstantial claim

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    Premisecitation

    CRISPRi knockdown is non-toxic in iPSCs and iPSC-derived cells, inducible, and reversible, enabling time-resolved analysis of gene function.

    “CRISPRi-based gene knockdown may result in weaker phenotypes than full gene knockout, however it is non-toxic in iPSCs and iPSC-derived cells76, inducible76 and reversible42, thereby enabling time-resolved analysis of gene function76.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [76] Potting, C. et al. Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy. Proc. Natl Acad. Sci. USA 115, E180–E189 (2018). source

      Not checked in the free run.

    • cites [42] Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). This paper describes the first platform for genome-wide CRISPRi and CRISPRa screens. source

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  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Understanding which gene expression changes are detrimental would prioritize those genes as therapeutic targets.

    “A detailed understanding of the genes expression changes of which are detrimental would prioritize these genes as potential therapeutic targets.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    CRISPRa assesses gain-of-function phenotypes and can complement CRISPR knockout or CRISPRi loss-of-function screens.

    “CRISPRa evaluates gain-of-function phenotypes and can provide complementary insight to CRISPR knockout or CRISPRi-based loss-of-function screens42.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). This paper describes the first platform for genome-wide CRISPRi and CRISPRa screens. source

      Not checked in the free run.

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

    Premisenone given

    Alpha-synuclein mutations can cause Parkinson's disease, in which substantia nigra dopaminergic neurons are selectively vulnerable while motor neurons are less vulnerable, despite widespread alpha-synuclein expression.

    “Conversely, mutations in alpha-synuclein can cause Parkinson’s Disease, where dopaminergic neurons in the substantia nigra are selectively vulnerable, but motor neurons are less vulnerable, even though alpha-synuclein is widely expressed in neurons.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Many neurological disorders, especially neurodegenerative diseases, are characterized by selective vulnerability of specific cell types.

  • May combine two claims.

    Interpretationreasoning

    Genetic screens directly identify targets and mechanisms, whereas small-molecule screens yield compounds whose targets and mechanisms are often hard to identify.

    “Second, while small-molecule screens yield compounds with beneficial effects, it is often a major challenge to identify their molecular targets and mechanism of action. By contrast, genetic screens directly identify relevant targets and mechanisms, for which effective compounds must then be developed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    GWAS often finds disease-associated variants outside protein-coding regions, and these can affect other genes' expression as eQTLs.

    “First, GWAS studies frequently uncover disease-associated genetic variants outside protein-coding regions. Such variants can affect the expression of other genes, thus acting as expression quantitative trait loci (eQTLs).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Neurons from familial and sporadic Alzheimer's patients show enlarged early endosomes, phosphorylated tau, and increased Aβ peptides and oligomers.

    “Neurons derived from familial as well as sporadic Alzheimer’s patient cells show enlarged early endosomes100, phosphorylated tau100, and increased production of Aβ peptides100 and oligomers101.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [100] Pasca, S. P. Assembling human brain organoids. Science 363, 126–127 (2019). source

      Not checked in the free run.

    • cites [101] Schwartz, M. P. et al. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proc. Natl Acad. Sci. USA 112, 12516–12521 (2015). source

      Not checked in the free run.

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

    Premisenone given

    CRISPRi and CRISPRa allow systematic control of specific gene expression levels in human cells.

    “CRISPRi and CRISPRa enable the systematic control of the expression levels of specific genes in human cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    CRISPR functional genomics allows specific gene perturbation, making causality establishment feasible.

    “CRISPR-based functional genomics enable the specific perturbation of genes of interests, thereby making it feasible to establish causality.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    iPSC-derived neurons resemble young neurons, while many neurological disorders affect older neurons, and aging is the main risk factor for neurodegenerative diseases.

    “Second, iPSC-derived neurons resemble young neurons, whereas many neurological disorders affect older neurons, and aging is the major risk factor for neurodegenerative diseases.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Aging is the major risk factor for neurodegenerative diseases.

  • May combine two claims.

    Premisecitation

    Directly reprogrammed fibroblasts retain some donor age features and aged neuron properties, but cannot be readily produced in quantities needed for large-scale screens.

    “Neurons directly reprogrammed from fibroblasts maintain some features reflecting the age of the donor and mirroring some properties of aged neurons120,121; however they are not readily obtained in quantities required for large-scale screens.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [120] Deczkowska, A. et al. Disease-associated microglia: a universal immune sensor of neurodegeneration. Cell 173, 1073–1081 (2018). source

      Not checked in the free run.

    • cites [121] Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). source

      Not checked in the free run.

48 of 48

  • The paper relies on this without support.

    Many neurodegenerative, neurodevelopmental, and neuropsychiatric disorders lack disease-modifying treatments.

    “Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, since many lack disease-modifying treatments.”

  • The paper relies on this without support.

    Limited understanding of causative molecular and cellular mechanisms is a major reason for the lack of effective therapies.

    “A major reason for this lack in effective therapies is our limited understanding of the causative molecular and cellular mechanisms.”

  • The paper relies on this without support.

    Many GWAS risk variants are located outside coding regions and are typically thought to affect expression of one or several coding genes.

    “Many risk variants identified in genome-wide association studies are located outside coding regions, and are typically thought to affect the expression of one or several coding genes.”

  • The paper relies on this without support.

    For many human genes, the molecular and cellular functions of the gene product are not well understood, and many genes have multiple functions, so genetic changes can have pleiotropic effects.

    “For many human genes, the molecular and cellular functions of the gene product are not well understood, and many genes have multiple functions – therefore genetic changes can have pleiotropic effects”

  • The paper relies on this without support.

    Selective vulnerability of specific cell types is a hallmark of many neurological diseases and drives disease-specific symptoms.

    “The selective vulnerability of specific cell types is a hallmark of many neurological diseases and drives disease-specific symptoms.”

  • The paper relies on this without support.

    Biopsies of the relevant nervous system tissues are generally not possible, so studies rely on post-mortem tissue.

    “However, biopsies of the relevant tissues of the nervous system are generally not possible, and studies rely on post-mortem tissue.”

  • The paper relies on this without support.

    After uptake, tau seeds need to escape the endolysosomal pathway to template aggregation of cytosolic tau.

    “After uptake, tau seeds need to escape the endolysosomal pathway to template aggregation of cytosolic tau.”

  • The paper relies on this without support.

    Nonhuman primates mirror humans more closely than mice in genome sequence, brain and circuit architecture, and have a complex behavioral and cognitive repertoire.

    “Nonhuman primates mirror humans much more closely in terms of their genome sequence, brain and circuit architecture than mice do, and they possess a complex behavioral and cognitive repertoire that facilitates evaluation of phenotypes relevant to neurological disease.”

  • The paper relies on this without support.

    Mouse models have advantages including relative ease of genetic manipulation, moderate cost, and the ability to study neuronal circuits and monitor behavioral and cognitive phenotypes.

    “Mice combine several advantages, including relative ease of genetic manipulation and moderate cost, the ability to study neuronal circuits and to monitor behavioral and cognitive phenotypes.”

  • The paper relies on this without support.

    Mouse models have provided important insights into neurological diseases and can serve as preclinical models to evaluate new therapeutic approaches.

    “They have provided important insights into neurological diseases, and can serve as pre-clinical models to evaluate new therapeutic approaches.”

  • The paper relies on this without support.

    Most academic laboratories lack the resources to perform large-scale arrayed screens.

    “most academic labs do not have the resources to conduct large-scale arrayed screens.”

  • The paper relies on this without support.

    Primary human nervous system cells are hard to obtain, and neurons are post-mitotic and cannot be expanded.

    “Primary human cells of the nervous system are difficult to obtain. Neurons in particular are post-mitotic and therefore non-expandable.”

  • The paper relies on this without support.

    Disease-associated genetic variants are being discovered rapidly, but their mechanisms are often unknown or controversial.

    “Genetic variants associated with familial disease or with disease risk in sporadic patients are being identified at a rapid pace. However, the mechanism by which these variants contribute to disease is often unknown or controversial.”

  • The paper relies on this without support.

    Many neurological disorders, especially neurodegenerative diseases, are characterized by selective vulnerability of specific cell types.

    “A hallmark of many neurological disorders, in particular neurodegenerative diseases, is the selective vulnerability of specific cell types.”

  • The paper relies on this without support.

    Genes usually act in combination with other genes rather than in isolation.

    “Genes typically do not function in isolation, but in combination with other genes.”

  • The paper relies on this without support.

    Most neurological diseases involve functional changes in specific nervous system cell types, and these functions can only be properly studied in those cell types.

    “First, most neurological diseases are characterized by changes in the function of specific cell types of the nervous system, and such functions can only be appropriately studied in the relevant cell types.”

  • The paper relies on this without support.

    Aging is the major risk factor for neurodegenerative diseases.

    “aging is the major risk factor for neurodegenerative diseases.”

  • The paper relies on this without support.

    The paper assumes that nearly all neurological disorders entail interactions among distinct cell types within a tissue environment.

    “most or all neurological disorders involve interactions between different cell types in a tissue context”

  • The paper relies on this without support.

    The paper assumes that neurological disorders appear as dysfunction of the nervous system and entail processes at the neuronal circuit level.

    “neurological diseases manifest as dysfunction of the nervous system, and involve processes at the level of neuronal circuits”

  • The paper relies on this without support.

    The paper assumes that clinical trials are presently ongoing for CRISPR-based treatments aimed at more accessible cell types, including blood cells and photoreceptor cells.

    “Clinical trials are currently underway for CRISPR-based therapies targeting cells that are more easily accessible, such as blood cells and photoreceptor cells.”
20 of 20

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The authors previously established a CRISPRi platform that enables robust knockdown of endogenous genes and large-scale loss-of-function genetic screens in human iPSC-derived neurons.

    “We previously established a CRISPRi platform that enables robust knockdown of endogenous genes and large-scale loss-of-function genetic screens in human iPSC-derived neurons3.”

    From Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis (2021) · cites this paper as [3]

  • The authors previously conducted a sub-genome scale CRISPRi screen targeting 2,325 druggable genome genes to reveal genes controlling neuronal survival.

    “We previously conducted a sub-genome scale CRISPRi screen to reveal genes controlling neuronal survival using an sgRNA library targeting 2,325 genes representing the “druggable genome”3.”

    From Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis (2021) · cites this paper as [3]

  • Knockdown of certain genes can have remarkably different impacts on cell survival and gene expression in different isogenic human cell types, including stem cells and neurons.

    “Cell-type specific gene function is also supported by our recent finding that knockdown of certain genes can have remarkably different impacts on cell survival and gene expression in different isogenic human cell types, including stem cells and neurons3.”

    From Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis (2021) · cites this paper as [3]

  • The authors previously developed a functional genomics platform enabling large-scale, multimodal loss-of-function genetic screens in differentiated human cell types, as demonstrated in neurons.

    “To this end, we recently developed a functional genomics platform, leveraging the strengths of CRISPR interference (CRISPRi) and induced pluripotent stem cell (iPSC) technology, that enables large-scale, multimodal loss-of-function genetic screens in differentiated human cell types, as demonstrated in neurons3.”

    From Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis (2021) · cites this paper as [3]

  • Genes involved in cholesterol biosynthesis were strongly enriched in CRISPRi hits with toxic knockdown phenotypes.

    “For example, genes involved in cholesterol biosynthesis were strongly enriched in hits with toxic knockdown phenotypes, suggesting an important role of cholesterol in maintaining neuronal survival, consistent with our previous findings3.”

    From Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis (2021) · cites this paper as [3]

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.

  • CRISPRa is described as both increasing and decreasing expression

    Minor

    The CRISPRa section says the method induces and overexpresses genes, but then calls its effect a knockdown. Those descriptions conflict.

  • RAB10 modifier citation points to a tau study

    Minor

    The text attaches reference 65 to the RAB10-APOE example, but entry 65 is about tau uptake, not that genetic interaction. The same sentence separately cites reference 85 for the RAB10 finding.

  • Insult-screen figure points to a genome-editing panel

    Minor

    The modifier-screen discussion directs readers to Fig. 2b for screens involving an insult. Earlier, the paper assigns Fig. 2b to deletions that cause gene loss of function; it assigns insult screens to Fig. 3b.

3 of 3

Cites

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Authors
Kampmann, M.
Affiliations
University of California, San Francisco (US)
Corresponding author
Kampmann, Martin
ORCID
Kampmann, Martin
Venue
Nature Reviews Neurology
Publisher
Springer Science and Business Media LLC
Published
2020-07-08
Volume
16
Issue
9
Pages
465-480
ISSN
  • 1759-4758 (ISSN-L)
  • 1759-4766
DOI
10.1038/s41582-020-0373-z
PMC
PMC7484261
OpenAlex
W3042084835
Cited by
163
References
142
Citations by year
2026: 24 · 2025: 32 · 2024: 29 · 2023: 21 · 2022: 28 · 2021: 26 · 2020: 3
FWCI
1.87
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Reviews Neurology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institutes of Health (1r56ag057528-01a1)
  • National Institute on Aging (R56 AG057528, R01AG062359)
  • National Institute of Mental Health (U01MH115747)
  • National Institute of General Medical Sciences (DP2GM119139)
  • National Institute of Neurological Disorders and Stroke (U54NS100717)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
neurological disorders, neurodegenerative diseases, neurodevelopmental disorders, neuropsychiatric disorders, CRISPR interference, CRISPR activation, genome-wide screen, induced pluripotent stem cells, brain organoids, disease-associated variants, genetic modifier screen, therapeutic target, disease mechanisms
MeSH terms
Gene Editing, Animals, Humans, Nervous System Diseases, Genomics, Genome-Wide Association Study, Induced Pluripotent Stem Cells, 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 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

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