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Cited by 322CRISPR and Genetic EngineeringNF-κB Signaling PathwaysCell Image Analysis Techniques

Optical Pooled Screens in Human Cells

Open access
Feldman, David, Singh, Avtar, Schmid-Burgk, Jonathan L., Carlson, Rebecca J., Mezger, Anja, Garrity, Anthony J., Zhang, Feng, Blainey, Paul C.·2019·Cell
DOI 10.1016/j.cell.2019.09.016PMC PMC6886477

Published version of a preprint

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Abstract

SUMMARY Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability, but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in NF-κB signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.

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Crossref · 10.1016/j.cell.2019.09.016

Cites

79 of 79 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 39 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Phenotype strength was well correlated between primary screening and validation ranks, with Spearman's ρ = 0.84 and 0.73 for IL-1β and TNFα, respectively.

    “Phenotype strength was well correlated between the primary screening and validation ranks (Spearman’s ρ = 0.84 and 0.73 for IL-1β and TNFα, respectively), emphasizing the quantitative nature of the primary screen (Figure S4B).”
    Quote in the paperSubstantial claim
  • Findingown data

    After serial transduction and antibiotic selection, 81% of cells contained reads mapping to two sgRNAs.

    “Most cells (81%) contained reads mapping to two sgRNAs (Figure S2C,D).”
    Quote in the paperSubstantial claim
  • Findingown data

    The in situ readout step can process millions of cells within a few days to provide high coverage (typically 100-1,000 cells/perturbation) needed for high-throughput optical screening.

    “We showed that the in situ readout step can process millions of cells within a few days to provide the high coverage (typically 100–1,000 cells/perturbation) needed for high-throughput optical screening.”
    Quote in the paperSubstantial claim
  • Findingown data

    Screening the same cell library by FACS with NGS readout showed similar enrichment of targeting sgRNAs, with 95% of barcodes within 5-fold abundance in both contexts.

    “Screening the same cell library by FACS with NGS readout showed similar enrichment of targeting sgRNAs (Figures 3C and S3D), with consistent representation of most barcodes in both contexts (95% within 5-fold abundance, Figure S1).”
    Quote in the paperSubstantial claim
  • Findingown data

    The paper reports an optical pooled screening method that expands the range of phenotypes amenable to large-scale pooled perturbation screens in mammalian cells.

    “Here, we report an optical pooled screening method that greatly expands the range of phenotypes amenable to large-scale pooled perturbation screens in mammalian cells.”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingreasoning

    In situ sequencing uses enzymatic amplification to generate high signal levels, permitting imaging at low magnification, which made it feasible to screen millions of cells from a pooled library.

    “In situ sequencing uses enzymatic amplification to generate high signal levels, permitting imaging at low magnification, which made it feasible for us to screen millions of cells from a pooled library.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The reporter is highly specific and sensitive, with mean in situ activation across 5 targeting sgRNAs of 65 ± 2.7% and background below 0.001% in the absence of a targeting sgRNA.

    “The reporter is highly specific and sensitive, with a mean in situ activation across 5 targeting sgRNAs of 65 ± 2.7% and background of <0.001 % in the absence of a targeting sgRNA (Figures 3B and S3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The CROP-seq vector directly sequencing sgRNA showed accurate sequencing in HeLa-TetR-Cas9 cells but reduced fluorescent signal intensity compared to LentiGuide-BC.

    “We observed accurate sequencing of sgRNAs in HeLa-TetR-Cas9 cells, but with reduced fluorescent signal intensity compared to LentiGuide-BC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hits included known pathway components annotated by KEGG for NF-κB activation by IL-1β signaling (5/5 genes), TNFα signaling (4/7 genes) and downstream components (5/7 genes).

    “The hits in our screen included known pathway components annotated by KEGG (Kanehisa and Goto, 2000) for NF-κB activation by IL-1β signaling (5/5 genes), TNFα signaling (4/7 genes) and downstream components (5/7 genes), including cytokine-specific receptors, adaptor proteins, and factors that activate the shared regulator MAP3K7 (Figure 4D) (Gewurz et al., 2012).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Kanehisa and Goto, 2000

      Not checked in the free run.

    • cites Gewurz et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Using the optimized protocol, in situ sequencing spots were readily visible at 10X magnification, with at least one exactly mapped read detected in more than 77% of transduced cells.

    “Using the optimized protocol, in situ sequencing spots were readily visible at 10X optical magnification, with at least one exactly mapped read detected in more than 77% of transduced cells (Figures 2B, 2D and 2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A total of 3,037,909 cells were retained for analysis after filtering based on reporter expression, nuclear morphology, and exact barcode mapping, with 952 out of 963 genes retained.

    “A total of 3,037,909 cells were retained for analysis after filtering cells based on reporter expression, nuclear morphology, and exact barcode mapping (952 out of 963 genes retained for analysis) (Figure S3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The pooled validation screen confirmed several genes initially detected as lower-ranked hits in the activation screen, while adding additional kinetic details.

    “The pooled validation screen confirmed several genes that were initially detected as lower-ranked hits in the activation screen, while adding additional kinetic details.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Members of the CUL1-RBX1-SKP1-FBXW11 complex and its substrate NEDD8 showed increased cell and nuclear area, with 4 out of 5 genes grouped together by PCA.

    “Members of the CUL1-RBX1-SKP1-FBXW11 complex and its substrate NEDD8 showed increased cell and nuclear area, with 4 out of 5 genes grouped together by PCA (Figures S4D-H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At later time points, RIPK1 showed an increased translocation defect while KCTD5 and HCFC1 were seen to negatively regulate p65.

    “For example, at later time points, RIPK1 showed an increased translocation defect while KCTD5 and HCFC1 were seen to negatively regulate p65.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    They optimized live-cell imaging and cell tracking with faster microscope hardware, linking time-resolved p65 translocation data to perturbation barcodes from 2,595,514 cells (median 3,611 cells/sgRNA) at 30 minute intervals.

    “We optimized conditions for live-cell imaging and cell tracking by using faster microscope hardware, allowing us to link time-resolved p65 translocation data to perturbation barcodes from 2,595,514 cells (median of 3,611 cells/sgRNA) at 30 minute intervals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    They performed a pooled validation screen for 61 genes with evidence for a kinetic phenotype, using 10 sgRNAs per gene.

    “Next, we performed a pooled validation screen for 61 genes that showed evidence for a kinetic phenotype (10 sgRNAs/gene).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The study shows that several perturbations can be identified in one cell, which offers a way to examine higher-order genetic interactions.

    “We demonstrate the identification of multiple perturbations within the same cell, providing a straightforward route to study higher order genetic interactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using a pooled live-cell imaging screen, the authors found two Mediator subunits that had not been known to negatively regulate pro-inflammatory signaling, and confirmed this in clonal knockouts.

    “Moreover, by performing a pooled live-cell imaging screen, we discovered two components of the Mediator complex as previously unknown negative regulators of pro-inflammatory signaling that we validated in clonal knockout cell lines.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Before this study, Mediator subunits were not known to act as negative regulators of NF-κB nuclear translocation or its transcriptional output.

  • May combine two claims.

    Findingnone given

    The 12-nucleotide barcodes in this work reliably separate over 80,000 perturbations, which is enough for genome-wide libraries.

    “The 12-nt sequences used here can robustly distinguish >80,000 perturbations, sufficient to encode genome-scale perturbation libraries.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Homozygous clonal knockouts of MED12 and MED24 each displayed delayed relaxation compared with wild-type cells.

    “Clonal homozygous knockouts of each of these Mediator complex subunits confirmed delayed relaxation relative to wild type cells (Figure 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Because the LentiGuide-BC and CROP-seq libraries produce mRNA that encodes each perturbation, the same cell populations can be analyzed in situ for dynamic or spatial readouts and also by methods like single-cell RNA-seq.

    “The LentiGuide-BC and CROP-seq libraries described in this study both generate mRNA encoding perturbation identity, so the same libraries of perturbed cells can be screened in situ to read dynamic and/or spatially resolved information and by complementary molecular profiling methods, such as single cell RNA-seq.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hits were identified at estimated false discovery rates below 10% and below 20% using the Benjamini-Hochberg procedure.

    “Hits at an estimated FDR <10% and <20% were identified using the Benjamini-Hochberg procedure (Table S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Rolling circle amplification allows rapid in situ sequencing with a low-power 10X objective, which raises throughput substantially.

    “Amplification by RCA enables fast in situ sequencing at low magnification (10X), greatly increasing throughput.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Knockout of negative regulators may lead to permanent baseline activation, causing negative feedback activation, potentially rendering cells refractory to induced NF-κB activation.

    “Knockout of these negative regulators may lead to permanent baseline activation of NF-κB signaling, which in turn causes negative feedback activation (Jäättelä et al., 1996), potentially rendering perturbed cells refractory to induced NF-κB activation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Jäättelä et al., 1996 source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Pooling gene perturbations increases scalability but cannot be combined with imaging of complex or dynamic cellular phenotypes.

    “Pooling gene perturbations greatly improves scalability, but is not compatible with imaging of complex and dynamic cellular phenotypes.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Gilbert et al., 2014 source

      Not checked in the free run.

    • cites Parnas et al., 2015 source

      Not checked in the free run.

    • cites Shalem et al., 2014 source

      Not checked in the free run.

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

    Premisecitation

    Historically pooled screens rely on enriching populations for a phenotype followed by NGS to measure perturbation abundance.

    “Historically, pooled screens have relied on enriching populations of cells for a phenotype of interest, followed by next-generation sequencing (NGS) to measure changes in perturbation abundance.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Shalem et al., 2014 source

      Not checked in the free run.

    • cites Wang et al., 2014 source

      Not checked in the free run.

    • cites Parnas et al., 2015 source

      Not checked in the free run.

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

    Premisecitation

    NF-κB is a family of transcription factors (p50, p52, p65, RelB, c-Rel) that translocate to the nucleus in response to stimuli.

    “After demonstrating the ability to screen genetic knockouts for optical phenotypes, we sought to identify genes required for activation of NF-κB, a family of transcription factors (p50, p52, p65, RelB and c-Rel) that translocate to the nucleus in response to a host of stimuli (Gewurz et al., 2012; Hayden and Ghosh, 2012; Pahl, 1999).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Gewurz et al., 2012 source

      Not checked in the free run.

    • cites Hayden and Ghosh, 2012 source

      Not checked in the free run.

    • cites Pahl, 1999 source

      Not checked in the free run.

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

    Premisecitation

    BAP1 has been previously described to deubiquitinate HCFC1, with relevance for controlling metabolism, ER-stress signaling, cell-cycle progression, and viral gene expression.

    “Among these, BAP1 has been previously described to deubiquitinate HCFC1 (Machida et al., 2009), with relevance for controlling metabolism (Bononi et al., 2017), ER-stress signaling (Dai et al., 2017), cell-cycle progression (Misaghi et al., 2009), and viral gene expression (Johnson et al., 1999).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Machida et al., 2009 source

      Not checked in the free run.

    • cites Bononi et al., 2017 source

      Not checked in the free run.

    • cites Dai et al., 2017 source

      Not checked in the free run.

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

    Premisecitation

    TAB1 knockdown was previously shown not to impact IKK activation.

    “Meanwhile, knockdown of TAB1 was previously shown not to impact IKK activation (Chen, 2005; Wang et al., 2001), consistent with our finding that TAB1 was not detected as a hit in any cell line.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Chen, 2005 source

      Not checked in the free run.

    • cites Wang et al., 2001 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Current CRISPR sgRNA libraries can be decoded directly via the CROP-seq vector, while other perturbations, such as ORFs or non-coding elements, can be tagged with short barcodes.

    “Existing CRISPR sgRNA libraries can be read out directly (using the CROP-seq vector) while other perturbations (e.g., ORFs, non-coding sequences) can be paired with short barcodes (Melnikov et al., 2012; Yang et al., 2011).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Melnikov et al., 2012 source

      Not checked in the free run.

    • cites Yang et al., 2011 source

      Not checked in the free run.

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

    Interpretationnone given

    Pooling raises data quality and cuts cost and labor sharply, so image-based phenotyping at genome scale becomes feasible for many labs.

    “Pooling improves data quality and dramatically reduces the cost and labor required, making genome-scale screens with image-based phenotyping accessible to many laboratories.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The translocation score was defined as the pixel-wise correlation between DAPI and p65-mNeonGreen within the nuclear region because this metric most clearly separated perturbations of known NF-κB genes from non-targeting controls.

    “Pixel-wise DAPI-mNeonGreen correlation within the segmented nuclear region, described above, was used to define the translocation score for each cell as it most clearly separated perturbations against known NF-κB genes from non-targeting controls.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Library representation was validated by next-generation sequencing.

    “Genomic DNA was also extracted for NGS validation of library representation.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    In situ sequencing can read any perturbation marked by a short expressed sequence, and it scales to millions of cells and genome-wide libraries.

    “The in situ sequencing framework is compatible with any perturbation that can be identified by a short expressed sequence and readily scales to millions of cells and genome-scale libraries.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The frameshift reporter screen was performed in three independent replicates.

    “This library was transduced into HeLa-TetR-Cas9-FR cells at MOI < 0.05 in three replicates, which were independently cultured and screened.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The chosen HeLa-TetR-Cas9 clone generated indels efficiently (97% or more) when doxycycline was present and cut little (2% or less) when it was absent.

    “The best clones showed efficient indel generation (≥ 97%) in the presence of doxycycline and minimal cutting (≤ 2%) in its absence.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Pooled genome-wide CRISPR screens that enrich cells by fitness or reporter fluorescence are now common in many systems, yet just one large CRISPR imaging screen, with 2,281 sgRNAs, has been published.

    “In recent years, there has been wide use of pooled genome-scale CRISPR screens based on enriching cells via fitness or reporter fluorescence in various model systems, but only one large-scale (2,281 sgRNA) CRISPR imaging screen (Groot et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Groot et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Protein barcodes based on epitopes offer a promising way to decode pooled elements without enzymes, but their current scale is only around 100 barcodes.

    “Epitope-based protein barcodes are a promising method for enzyme-free decoding of pooled elements, but are currently limited in scale to ~100 barcodes (Wroblewska et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wroblewska et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Using the second-largest sgRNA translocation defect as the gene-level defect reduces false positives from clonal effects and false negatives from inefficient sgRNAs.

    “This statistic helps reduce the false positive rate due to clonal effects (integration of an sgRNA into a cell that is defective in translocation) which are independent among sgRNAs and screen replicates, as well as false negatives due to inefficient sgRNAs.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Clonal effects from integration of an sgRNA into a translocation-defective cell are independent across sgRNAs and screen replicates.

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

    Premisecitation

    Pro-IL-1β is a known NF-κB target during Toll-like receptor activation.

    “pro-IL-1β, which is a well-established NF-κB target gene in the context of Toll-like receptor activation (Sims and Smith, 2010)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sims and Smith, 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    The approach is broadly applicable across many settings.

    “Our approach is broadly applicable across many settings.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The primary optical screen is highly sensitive, as shown by detecting the weaker phenotype of a mono-allelic MED24 knockout despite Cas9-generated allele heterogeneity.

    “underscoring the high sensitivity of the primary optical screen despite the heterogeneity of alleles generated by Cas9 cleavage (Figure 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Optical pooled screening is a useful counterpart to single-cell molecular profiling, which yields rich data but still lacks dynamic and spatial resolution.

    “Optical pooled screening serves as an important complement to single-cell molecular profiling, which also provides high-content data but is not yet able to deliver dynamic or spatially resolved information.”
    Quote in the paperSubstantial claim
38 of 38

  • The paper relies on this without support.

    Pooled libraries can be constructed, introduced, and read out as single samples, reducing cost, effort, and time for large-scale screens.

    “Pooled libraries can be constructed, introduced into cells, and read out as single samples, dramatically reducing the cost, effort and time needed to perform large-scale screens.”

  • The paper relies on this without support.

    Combining all perturbations into one sample reduces batch effects and improves statistical power because all cells experience the same conditions.

    “Moreover, combining all perturbations into a single sample reduces batch effects and improves statistical power since all perturbed and control cells experience the same conditions.”

  • The paper relies on this without support.

    Enrichment-based pooled screens are limited to population-level measurement, often rely on indirect reporters, and limit phenotypes to one or few parameters.

    “Although highly scalable, these approaches are limited to population-level measurement, often rely on indirect reporters for the biological activity of interest, and necessarily limit phenotypes to one or a few parameters.”

  • The paper relies on this without support.

    Existing single-cell molecular profiling approaches are destructive and cannot directly monitor dynamic processes over time in individual cells.

    “Moreover, existing single cell molecular profiling approaches are destructive and thus cannot be used to directly monitor dynamic processes over time in individual cells.”

  • The paper relies on this without support.

    p65 nuclear translocation is a valid measure of NF-κB activation.

    “We used an established nuclear translocation assay to measure the localization of a p65-mNeonGreen reporter in HeLa cells following stimulation with either IL-1β or TNFα, cytokines that activate NF-κB via different pathways (Figure 4A).”

  • The paper relies on this without support.

    At baseline, NF-κB dimers are held inactive in the cytoplasm by IκB proteins that mask nuclear localization signals.

    “At baseline, NF-κB dimers are maintained in an inactive state in the cytoplasm by inhibitory IκB proteins, which mask nuclear localization signals to prevent NF-κB translocation into the nucleus.”

  • The authors state this as an assumption.

    Ubiquitin signaling may have unrecognized roles in NF-κB activation and relaxation.

    “We included all GO-annotated ubiquitin ligase and deubiquitinase enzymes, as well as 425 immune-related genes in the library, hypothesizing that ubiquitin signaling may play as-yet unrecognized roles in NF-κB activation and relaxation (return to the baseline cellular distribution) (Chen, 2005) (Figure 4A, Table S3).”

  • The paper relies on this without support.

    The kinetics of NF-κB signaling events and feedback loops determine the pathway's dynamic response.

    “Activation of NF-κB involves a cascade of signaling events and feedback loops whose kinetics determine the dynamic response of the pathway.”

  • The paper relies on this without support.

    Before this study, Mediator subunits were not known to act as negative regulators of NF-κB nuclear translocation or its transcriptional output.

    “Mediator components have not previously been shown to be involved in negative regulation of either the nuclear translocation or the transcriptional response of NF-κB.”

  • The paper relies on this without support.

    Secretion of IL-1β requires both NF-κB-driven transcription and Caspase 1-mediated post-transcriptional processing.

    “As one of the most potent pyrogens in the human body, IL-1β is secreted only upon transcriptional (NF-κB) as well as post-transcriptional (Caspase 1) licensing.”

  • The paper relies on this without support.

    Large-scale arrayed perturbation screens are so costly, slow, and laborious that optical phenotyping has seen little use in screening.

    “Despite the rich phenotypic information provided by imaging assays, optical phenotyping has been underused for screening applications due to the substantial cost, time and labor required to execute large-scale arrayed perturbation screens.”

  • The paper relies on this without support.

    Published highly multiplexed FISH approaches require 60X imaging and barcodes over 200 bp, so direct oligo array synthesis is not cost-effective.

    “By comparison, reported methods for highly multiplexed FISH require higher imaging magnification (60X) and barcodes longer than 200 bp, precluding cost-effective direct oligo array synthesis.”

  • The authors state this as an assumption.

    All HA+ cells that mapped to a non-targeting control sgRNA, which was 4.7% of such cells, were false positives caused by incorrect barcode assignment.

    “we first assumed all HA+ cells mapped to a non-targeting control sgRNA (4.7%) were false positive events due to incorrect barcode assignment”

  • The paper relies on this without support.

    Incorrect barcode assignments were equally likely to map an HA+ cell to a targeting sgRNA or to a control sgRNA.

    “as incorrect barcode assignments were equally likely to map an HA+ cell to a targeting or control sgRNA”

  • The paper relies on this without support.

    Clonal effects from integration of an sgRNA into a translocation-defective cell are independent across sgRNAs and screen replicates.

    “which are independent among sgRNAs and screen replicates”
15 of 15

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?

  • Next-generation sequencing approaches have been adapted to enable in situ sequencing of DNA barcodes tagging imaged cells.

    “In recent years, next-generation sequencing approaches have been adapted to enable in situ sequencing of DNA barcodes tagging imaged cells (e.g. (94, 95)).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [95]

  • Some biological processes are better assayed using alternative modalities (splicing, chromatin, protein, imaging).

    “In addition, some biological processes are better assayed using alternative modalities, including mRNA splicing, chromatin state, protein level, and imaging, all of which are now being studied at scale with single cell CRISPR screens39-44.”

    From Transcriptome-wide analysis of differential expression in perturbation atlases (2025) · cites this paper as [40]

  • Human genetics has successfully identified genetic modifiers for neurodegeneration, such as RAB10 for APOE in Alzheimer's disease and TMEM106B for GRN in frontotemporal dementia.

    “Genetic modifiers for neurodegeneration have been identified successfully through human genetics, such as RAB10 for APOE65 in Alzheimer’s Disease (variants in the RAB10 locus are linked to resilience in individuals carrying the high-risk APOE ε4 allele85), and TMEM106B for GRN66, 67 in frontotemporal dementia (SNPs affecting TMEM106B expression are protective in carriers of disease-associated GRN”

    From CRISPR-based functional genomics for neurological disease (2020) · cites this paper as [85]

  • Combining functional methods with complex readouts can generate empirical truth data that supports in silico models predicting causal variants, effector transcripts, and cellular effects.

    “Combined with complex readouts–including high-content imaging128 and single-cell transcriptomics and epigenomics129,130–these methods can generate empirical ‘truth’ data, supporting the development of in silico models to predict causal variants, effector transcripts126 and cellular effects.”

    From A brief history of human disease genetics (2020) · cites this paper as [128]

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.

  • Validation screen has two reported gene counts

    Minor

    The Results describe the pooled validation screen as covering 61 genes, but the Methods say its library targeted 65 candidate genes. The text does not state whether four genes in that library were excluded from the reported analysis.

1 of 1

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Authors
Feldman, D., Singh, A., Schmid-Burgk, J. L., Carlson, R. J., Mezger, A., Garrity, A. J., Zhang, F., & Blainey, P. C.
Affiliations
Broad Institute (US)
Corresponding author
Blainey, Paul C.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2019-10
Volume
179
Issue
3
Pages
787-799.e17
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2019.09.016
PMC
PMC6886477
OpenAlex
W2981110120
Semantic Scholar
4ac904487e165a54a99e56a0af9b952e502302a2
Cited by
321
References
79
Citations by year
2026: 49 · 2025: 68 · 2024: 53 · 2023: 37 · 2022: 39 · 2021: 30 · 2020: 33 · 2019: 12
FWCI
9.43
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
Funders
National Human Genome Research Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Keywords
demultiplexing, image-based phenotyping, NF-κB signaling, Mediator complex, live-cell imaging, mammalian cells
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Animals, Cell Line, Cell Nucleus, Genetic Testing, Humans, NF-kappa B, Genomics, Transcription Factor RelA, Mediator Complex, CRISPR-Cas Systems

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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.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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