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Cited by 556CRISPR and Genetic EngineeringPluripotent Stem Cells ResearchVirus-based gene therapy research

CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons

Open access
Tian, Ruilin, Gachechiladze, Mariam A., Ludwig, Connor H., Laurie, Matthew T., Hong, Jason Y., Nathaniel, Diane, Prabhu, Anika V., Fernandopulle, Michael S. +4 more·2019·Neuron
DOI 10.1016/j.neuron.2019.07.014PMC PMC6813890

Published version of a preprint

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Abstract

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.

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Crossref · 10.1016/j.neuron.2019.07.014

Cites

67 of 67 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 20 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    CRISPRi produces strong and lasting reduction of endogenous gene expression in human neurons made from iPSCs.

    “We demonstrate robust and durable knockdown of endogenous genes in such neurons”
    • Also stated in: § Robust CRISPR interference in human iPSC-derived neurons · boosted
      “Our platform thus enables potent CRISPRi knockdown of endogenous genes in iPSC-derived neurons.”
    Quote in the paperSubstantial claim
  • Findingown data

    Expression of the CRISPRi machinery and/or sgRNAs did not affect neuronal activity measured by calcium imaging.

    “or activity as evaluated by calcium imaging (Fig. S1F and Movies S1, S2).”
    Quote in the paperSubstantial claim
  • Findingown data

    Expression of the CRISPRi machinery and/or sgRNAs did not produce detectable DNA damage in iPSCs.

    “We found that expression of CRISPRi machinery and/or sgRNAs did not cause detectable DNA damage (Fig. S1C,D)”
    Quote in the paperSubstantial claim
  • Findingown data

    The vast majority of sgRNAs that altered survival in pooled screens also altered survival in the arrayed longitudinal survival analysis.

    “the vast majority of sgRNAs that altered survival in pooled screens also altered survival in our arrayed longitudinal survival analysis (Fig. 7D).”
    Quote in the paperSubstantial claim
  • Findingown data

    The vast majority of neuron-essential genes were detectable at the transcript level.

    “The vast majority of neuron-essential genes were detectable at the transcript level, further supporting the specificity of our screen results.”
    Quote in the paperSubstantial claim
  • May combine two claims. 20
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    Expression of the CRISPRi machinery and/or sgRNAs did not affect neuronal differentiation.

    “and did not affect neuronal differentiation (Fig. S1E)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The screen identified a shared core set of essential genes plus additional iPSC-specific and neuron-specific essential genes.

    “This analysis revealed a shared core set of essential genes, as expected, and additional iPSC-specific and neuron-specific essential genes (Fig. 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only a few genes showed high similarity in transcriptomic changes between iPSCs and neurons; most genes induced distinct responses.

    “only a few genes, including SQLE, MMAB, MVK, UQCRQ, and ATP5B, showed high similarity (similarity score > 0.15) in the transcriptomic changes they induced in iPSCs versus neurons. Knockdown of most genes induced distinct transcriptomic responses in the two cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MAT2A is essential in neurons but not iPSCs.

    “MAT2A is essential in neurons, but not iPSCs (Fig. 6B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    sgRNAs targeting PPP1R12C were strongly enriched when TMP was added at the iPSC stage, but this phenotype was substantially weaker when TMP was added at the neuron stage.

    “sgRNAs targeting PPP1R12C were strongly enriched when TMP was added at the iPSC stage, but this phenotype was substantially weaker when TMP was added at the neuron stage.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MAP3K12 knockdown caused downregulation of many cytoskeletal organization proteins and upregulation of specific synaptotagmins.

    “MAP3K12 knockdown caused downregulation of a vast array of proteins involved in cytoskeletal organization, and upregulation of specific synaptotagmins”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MAP3K12 knockdown in neurons caused upregulation of ribosomal genes and POU4F1.

    “Ribosomal genes and the anti-apoptotic transcription factor Brn3a (encoded by POU4F1) were upregulated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Knockdown phenotypes of hit genes were strongly correlated between neurons at different time points but less correlated between neurons and iPSCs.

    “Knockdown phenotypes of hit genes were strongly correlated between neurons at different time points, but distinctly less correlated between neurons and iPSCs (Fig. 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All positive hits and most negative hits from the primary screen were confirmed in the pooled validation screen in iPSCs and neurons.

    “In both iPSCs and neurons, all positive hits and most of the negative hits from the primary screen were confirmed in the validation screen (Fig. 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither CRISPRi machinery nor non-targeting sgRNAs affected neurite growth.

    “or neurite growth (Fig. S6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither CRISPRi machinery nor non-targeting sgRNAs affected neuronal survival.

    “We found that neither CRISPRi machinery nor non-targeting sgRNAs affected neuronal survival (Fig. S6B)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    UBA1 is essential in both neurons and iPSCs.

    “knockdown of the essential E1 ubiquitin activating enzyme, UBA1 (Fig. 5B)”
    Quote in the paperSubstantial claim

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

    Findingown data

    The median number of genes detected per cell in the CROP-Seq dataset was approximately 5,000 for iPSCs and 4,600 for neurons.

    “Median number of genes detected per cell was around 5,000 for iPSCs and 4,600 for neurons.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The mean reads per cell in the CROP-Seq dataset were approximately 84,000 for iPSCs and 91,000 for neurons.

    “The mean reads per cell was around 84,000 for iPSCs and 91,000 for neurons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The i3Neuron platform yields large numbers of highly uniform neurons, which is necessary for robust population-based screens.

    “which yields large quantities of highly homogeneous neurons, a prerequisite for robust population-based screens.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Fernandopulle et al., 2018 source

      Not checked in the free run.

    • cites Wang et al., 2017 source

      Not checked in the free run.

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

    Premisecitation

    Induced expression of different transcription factors yields other neuron types, such as motor neurons and inhibitory neurons.

    “induced expression of different transcription factors yield other types of neurons, such as motor neurons (Hester et al., 2011; Shi et al., 2018) and inhibitory neurons (Yang et al., 2017).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hester et al., 2011 source

      Not checked in the free run.

    • cites Shi et al., 2018 source

      Not checked in the free run.

    • cites Yang et al., 2017 source

      Not checked in the free run.

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

    Interpretationnone given

    iPSC-derived neurons do not fully recapitulate all features of mature or aging neurons in the human brain.

    “Despite their usefulness, iPSC-derived neurons have limitations – in particular, they do not fully recapitulate all features of mature (or aging) neurons in the human brain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    CRISPR interference is inducible and reversible.

    “Second, it is inducible and reversible (Gilbert et al., 2014)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Gilbert et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The large-scale CRISPRi screen in human iPSC-derived neurons uncovered genes controlling neuronal survival but not cancer cell or iPSC survival.

    “our large-scale CRISPRi screen in human iPSC-derived neurons uncovered genes that control the survival of neurons, but not cancer cells or iPSCs”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationnone given

    Neurons are a highly specialized and disease-relevant cell type, so certain human gene functions must be studied in these cells.

    “Neurons are a highly specialized and disease-relevant cell type, and thus it is crucial to study certain human gene functions in these cells.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Ngn2-driven generation yields glutamatergic neurons.

    “In addition to the Ngn2-driven generation of glutamatergic neurons (Fernandopulle et al., 2018; Wang et al., 2017; Zhang et al., 2013) used here”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Fernandopulle et al., 2018 source

      Not checked in the free run.

    • cites Wang et al., 2017 source

      Not checked in the free run.

    • cites Zhang et al., 2013 source

      Not checked in the free run.

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

    Study design and conductown data

    In the pooled validation screen, cells were transduced at 70% MOI and selected to obtain a population that was approximately 85% BFP-positive.

    “CRISPRi-i3N iPSCs were transduced with the pool at 70% MOI (quantified as fraction of BFP-positive cells by flow cytometry) and were transduced cells selected by 1 ug/ml of puromycin to obtain a population of cells that was ~85% BFP-positive.”
    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 conductown data

    In the pooled validation screen, mono-culture neuronal samples had library representations of approximately 35,000 cells per library element on Day 14 and approximately 28,000 on Day 28.

    “the library representation was ~35,000 cells / library element on Day 14 and ~28,000 cells / library element on Day 28.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the CROP-Seq analysis, an sgRNA was assigned to a cell only if its UMI count exceeded 4 standard deviations above the mean UMI count of all sgRNAs.

    “For a given cell, sgRNA(s) whose UMI counts were greater than 4 standard deviations of the mean UMI counts of all sgRNAs were assigned to that cell as its identity.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    In the pooled validation screen, the Day 0 sample had a library representation of approximately 9,000 cells per library element.

    “approximately 2 million of these cells were harvested as Day 0 sample (corresponding to a library representation of ~9,000 cells/library element)”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the CROP-Seq analysis, only cells with a single assigned sgRNA were retained for further analysis.

    “Cells with only one assigned sgRNA were retained for further analysis.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    In the longitudinal imaging screen, a gene targeted by two sgRNAs was discarded if the correlation between the two sgRNAs was less than 0.8.

    “a gene was discarded if it was targeted by two sgRNAs and the correlation of the two sgRNAs was less than 0.8.”
    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 conductown data

    The primary CRISPRi screen achieved an initial MOI of approximately 15%, corresponding to a library representation of approximately 450 cells per library element.

    “The initial MOI, quantified as the fraction of BFP-positive cells by flow cytometry, was ~15%, corresponding to a library representation of ~450 cells per library element.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    In the pooled validation screen, co-culture neuronal Day 14 samples had a library representation of approximately 50,000 neurons per library element.

    “Day 14 neurons from each replicate of co-culture experiment were harvested, the library representation was ~50,000 neurons/library element.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    In the pooled validation screen, Day 10 iPSC samples had a library representation of approximately 9,000 cells per library element.

    “Approximately 2 million of Day 10 iPSCs from each replicate were harvested, corresponding to a library representation of ~9,000 cells/library element.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    CROP-Seq was performed at a low multiplicity of infection to minimize double infection.

    “CRISPRi-i3 N iPSCs were infected with a pool of selected sgRNAs in the CROP-Seq vector pMK1334 at a low multiplicity of infection to minimize double infection.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The 192 sgRNAs used in the pooled validation screen were verified by Sanger sequencing.

    “192 sgRNAs, including 184 sgRNAs targeting 92 selected hit genes from the primary screen (two sgRNAs per gene) and 8 non-targeting control sgRNAs, were individually cloned into the secondary screening vector pMK1334 and verified by Sanger sequencing.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    In the neuronal survival screen, estimated library representation was approximately 410 cells per library element on Day 14, approximately 380 on Day 21, and approximately 330 on Day 28.

    “the estimated library representation for these time points was ~410 cells/library element on Day 14, ~380 cells/library element on Day 21, and ~330 cells/library element on Day 28.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For each target gene in the CROP-Seq analysis, only the top 50% of cells with the best on-target knockdown were retained.

    “For each target gene, the top 50% cells with best on-target knockdown were retained.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    In the pooled validation screen, sgRNA-level knockdown phenotype scores were normalized by subtracting the median from non-targeting control sgRNAs.

    “Subsequently, knockdown phenotype scores for each sgRNA were calculated as LFCs of sgRNA counts between two samples and were normalized by subtracting the median of non-targeting control sgRNAs.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    After puromycin treatment in the primary screen, 83% of cells expressed high levels of BFP.

    “At the end of treatment, cells were assessed by flow cytometry (83% expressed high levels of BFP)”
    Quote in the paperNot a substantial claim
36 of 36

  • The paper relies on this without support.

    The AAVS1 locus, where the inducible Ngn2 transgene was integrated, lies within the PPP1R12C gene.

    “Interestingly, the AAVS1 locus, into which the inducible Ngn2 transgene was integrated, resides within the PPP1R12C gene.”

  • The paper relies on this without support.

    Primary human neurons are difficult to obtain from human donors and cannot be expanded because they are post-mitotic.

    “primary neurons are difficult to obtain from human donors, and non-expandable due to their post-mitotic nature.”

  • The paper relies on this without support.

    Most previous CRISPR-based screens were conducted in cancer cell lines or stem cells rather than healthy differentiated human cells.

    “Most previous CRISPR-based screens were implemented in cancer cell lines or stem cells rather than healthy differentiated human cells”

  • The paper relies on this without support.

    CRISPRi reduces gene function by partial knockdown rather than complete knockout, allowing study of essential genes.

    “Furthermore, CRISPRi perturbs gene function by partial knockdown, rather than knockout, thereby enabling the investigation of the biology of essential genes.”

  • The paper relies on this without support.

    sgRNA abundance in pooled screens reflects cell survival or proliferation.

    “Based on the depletion or enrichment of sgRNAs targeting specific genes at different time points compared to day 0, we identified hit genes for which knockdown was toxic or beneficial to either iPSCs or neurons at different time points (Fig. 2B, Fig. S2B).”

  • The paper relies on this without support.

    iPSCs can be made from readily available cells, genetically engineered, expanded, and differentiated into various cell types.

    “They can be made from readily available cells, such as skin fibroblasts or peripheral mononuclear blood cells; they can be genetically engineered and subsequently expanded to generate large numbers of isogenic cells; and they can then be differentiated into a variety of cell types”

  • The paper relies on this without support.

    The presence of astrocytes in co-culture is representative of the brain environment.

    “In the brain, many neuronal functions are supported by glial cells, particularly astrocytes.”

  • The paper relies on this without support.

    CRISPR interference reduces gene expression partially rather than completely eliminating it.

    “Third, it perturbs gene function via partial knockdown, as opposed to knockout”
8 of 8

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?

  • 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.”

    From CRISPR-based functional genomics for neurological disease (2020) · cites this paper as [82]

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.

  • Doxycycline concentration differs by a factor of 1,000

    Minor

    The neuronal-plating procedures specify 2 μg/μL doxycycline in Cortical Neuron Culture Medium, whereas the formulation of that same medium specifies 2 μg/mL.

1 of 1

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Authors
Tian, R., Gachechiladze, M. A., Ludwig, C. H., Laurie, M. T., Hong, J. Y., Nathaniel, D., Prabhu, A. V., Fernandopulle, M. S., Patel, R., Abshari, M., Ward, M. E., & Kampmann, M.
Affiliations
  • University of California, San Francisco (US)
  • National Institutes of Health (US)
Corresponding author
  • Ward, Michael E.
  • Kampmann, Martin
ORCID
Venue
Neuron
Publisher
Elsevier BV
Published
2019-10
Volume
104
Issue
2
Pages
239-255.e12
ISSN
0896-6273 (ISSN-L)
DOI
10.1016/j.neuron.2019.07.014
PMC
PMC6813890
OpenAlex
W2967789674
Cited by
543
References
68
Citations by year
2026: 85 · 2025: 116 · 2024: 103 · 2023: 60 · 2022: 66 · 2021: 62 · 2020: 45 · 2019: 6
FWCI
15.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Neuron (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institute of General Medical Sciences
  • Paul G. Allen Family Foundation
  • National Institute of Neurological Disorders and Stroke
  • National Institute on Aging
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
CRISPR interference, iPSC-derived neurons, functional genetic screens, gene knockdown, neuronal survival, essential genes, single-cell transcriptomics, neuronal morphology
MeSH terms
RNA-Seq, Cell Survival, Humans, Neurons, Microscopy, Confocal, Gene Knockdown Techniques, Induced Pluripotent Stem Cells, Single-Cell Analysis, CRISPR-Cas Systems

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