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Cited by 573CRISPR and Genetic EngineeringCAR-T cell therapy researchT-cell and B-cell Immunology

Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function

Open access
Shifrut, Eric, Carnevale, Julia, Tobin, Victoria, Roth, Theodore L., Woo, Jonathan M., Bui, Christina T., Li, P. Jonathan, Diolaiti, Morgan E. +2 more·2018·Cell
DOI 10.1016/j.cell.2018.10.024PMC PMC6689405

Published version of a preprint

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Abstract

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2018.10.024

Cites

83 of 83 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 32 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    At day 4 after electroporation, more than 80% of transduced (mCherry+) cells were CD8 negative following CD8A targeting.

    “At day 4 post-electroporation, the transduced cells (mCherry+) were largely (>80%) CD8 negative (Figure 1B and Figure S1E), indicative of successful targeting by the Cas9-sgRNA combination.”
    Quote in the paperSubstantial claim
  • Findingown data

    Sorting dividing and non-dividing primary cells by CFSE gave stronger sgRNA enrichment than growth-based screens with otherwise identical timelines.

    “Importantly, sorting dividing and non-dividing primary cells based on CFSE provided stronger enrichment of sgRNA sequences than simple growth-based screens with otherwise identical experimental timelines (Figure S1J).”
    Quote in the paperSubstantial claim
  • Findingown data

    SLICE pooled CRISPR screens can discover both positive and negative regulators of proliferation in primary human T cells.

    “Taken together, these data demonstrate that SLICE pooled CRISPR screens can be used to discover positive and negative regulators of proliferation in primary human T cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines.
    • Rests on an assumption: Primary human T cells can only be cultured ex vivo for limited time spans.
    • Rests on an assumption: Current pooled CRISPR screening methods rely on establishing cell lines with stably integrated Cas9 expression cassettes.
  • Findingcitation

    Targeting CD5 or CBLB enhanced proliferation in human T cells.

    “We also found that proliferation could be enhanced in human T cells by targeting CD5 or CBLB, which have reported roles in the negative regulation of T cell stimulation responses (Voisinne et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Voisinne et al., 2016 source

      Not checked in the free run.

  • Findingown data

    The primary and secondary genome-wide screens were well correlated.

    “The results were well correlated between the primary and secondary screens (Figure 2C).”
    Quote in the paperSubstantial claim
  • May combine two claims. 29
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 15

  • May combine two claims.

    Findingown data

    Targeting PTPRC (CD45) confirmed successful knockout at a second target and showed efficacy in both CD8+ and CD4+ T cells.

    “By targeting PTPRC (CD45) with the same delivery strategy, we confirmed successful knockout at a second target and demonstrated efficacy of the system in both CD8+ and CD4+ T cells (Figure S1F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Loss of CD8 protein was specifically caused by the targeting sgRNA, since cells with a non-targeting control sgRNA retained high CD8 expression.

    “Loss of CD8 protein was specifically programmed by the targeting sgRNA, as cells transduced with a non-targeting control sgRNA retained high levels of CD8 expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Genome-wide loss-of-function screens in primary human T cells identified essential TCR signaling components and genes that negatively tune proliferation after stimulation.

    “Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines.
    • Rests on an assumption: Primary human T cells can only be cultured ex vivo for limited time spans.
    • Rests on an assumption: Current pooled CRISPR screening methods rely on establishing cell lines with stably integrated Cas9 expression cassettes.

  • May combine two claims.

    Findingown data

    SLICE (sgRNA lentiviral infection with Cas9 protein electroporation) achieves effective and specific disruption of target genes in primary human T cells.

    “We conclude that sgRNA lentiviral infection with Cas9 protein electroporation (SLICE) results in effective and specific disruption of target genes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines.
    • Rests on an assumption: Primary human T cells can only be cultured ex vivo for limited time spans.
    • Rests on an assumption: Current pooled CRISPR screening methods rely on establishing cell lines with stably integrated Cas9 expression cassettes.

  • May combine two claims.

    Findingown data

    Unstimulated T cells had donor-dependent basal gene expression patterns, while stimulated cells from the two donors tended to share a transcriptional signature and cluster together.

    “While the unstimulated T cells had donor-dependent basal gene expression patterns, stimulated cells from the two donors tended to have a shared transcriptional signature and clustered together.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    sgRNAs targeting CD3D or LCP2 inhibited the cluster 10 activation program and promoted the cluster 1-2 programs, characterized by expression of IL7R and CCR7.

    “In contrast, sgRNAs targeting CD3D or LCP2 inhibited the cluster 10 activation program and promoted the cluster 1–2 programs, characterized by expression of IL7R and CCR7.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gene targets that impaired TCR-dependent proliferation included those encoding the delta and zeta chains of the TCR complex (negative ranks 18 and 6), LCK (negative rank 20), and ZAP70 (negative rank 299).

    “gene targets that impaired TCR dependent proliferation included those encoding delta and zeta chains of the TCR complex itself (negative rank 18 and 6, respectively), and LCK (negative rank 20) which directly phosphorylates and activates the TCR ITAMs and the central signaling mediator ZAP70 (negative rank 299) (Dave et al., 1998; Tsuchihashi et al., 2000; Wang et al., 2010).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Dave et al., 1998 source

      Not checked in the free run.

    • cites Tsuchihashi et al., 2000 source

      Not checked in the free run.

    • cites Wang et al., 2010 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Cells with non-targeting control sgRNAs were relatively evenly distributed among clusters, while cells with CBLB and CD5 sgRNAs were enriched in clusters associated with proliferation and activation, and cells with LCP2 sgRNAs were mostly in clusters characterized by resting profiles.

    “Cells with non-targeting control sgRNAs were relatively evenly distributed among clusters. In contrast, cells with CBLB and CD5 sgRNAs were enriched in clusters associated with proliferation and activation, and those with LCP2 sgRNAs were found mostly in clusters characterized by resting profiles (Figure 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CBLB and CD5 knockout cells showed a marked increase in divisions after stimulation compared to controls, persistent across different guide RNAs and blood donors.

    “For example, CBLB and CD5 knockout cells showed a marked increase in number of divisions post stimulation compared to controls, persistent across different guide RNAs and blood donors (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percent of cells expressing activation markers was higher for positive hits than for non-targeting control guides, consistent across two sgRNAs per gene for four donors.

    “Overall, the percent of cells expressing these activation markers in each condition was higher for positive hits compared to non-targeting control guides, consistent across two sgRNAs per gene, for four donors (Figure 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TCR stimulation had a strong effect in inducing an activated cell state across biological replicates, although more cells appeared strongly stimulated in Donor 1 than in Donor 2.

    “TCR stimulation thus had a strong effect in inducing an activated cell state across biological replicates, although more cells appeared to have been stimulated strongly in Donor 1 than in Donor 2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    NY-ESO TCR-transduced T cells triggered caspase-dependent death of A375 targets, evidenced by a caspase increase and a time-dependent drop in RFP-tagged A375 nuclei.

    “These transduced T cells were able to induce caspase-mediated cell death in the target A375 cells, which was exhibited by a rise in the level of caspase and a decline in the level of RFP-tagged A375 nuclei over time (Figure S5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    UBASH3A, CBLB, CD5, and RASA2 knockout T cells all showed greater than 2-fold increase in proliferation index compared to non-targeting control cells.

    “UBASH3A, CBLB, CD5, and RASA2 knockout T cells all showed greater than 2-fold increase in the proliferation index compared to non-targeting control cells (Figure 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SLICE genome-wide screening was adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling.

    “SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ablating TCEB2, SOCS1, CBLB, or RASA2 each led to significantly greater tumor cell clearance than non-targeting control T cells.

    “In contrast, CRISPR-ablation of negative regulators TCEB2, SOCS1, CBLB and RASA2 each significantly increased tumor cell clearance compared to control T cells electroporated with a non-targeting guide RNA (Figure 5C).”
    • Also stated in: § Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells · plain
      “Targeted deletion of these four genes led to improved kinetics of cancer cell clearance in our assay compared to the non-targeting control conditions (Figure 5D and Figure S5C–D).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    Multiple sgRNAs targeting CD5 or CBLB had concordant effects, indicating the phenotype was not due to off-target effects.

    “Furthermore, multiple sgRNAs targeting these genes had concordant effects, increasing our confidence that the phenotype was not due to off-target effects (Figure S1I, Table S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    TCEB2 (rank 5) complexes with RNF7 (rank 34), CUL5 (rank 162), and SOCS1 (rank 3), a key suppressor of JAK/STAT signaling in activated T cells.

    “TCEB2 (rank 5) complexes with RNF7 (rank 34), CUL5 (rank 162), and SOCS1 (rank 3), a key suppressor of JAK/STAT signaling in activated T cells (Kamura et al., 1998; Liau et al., 2018).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kamura et al., 1998 source

      Not checked in the free run.

    • cites Liau et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    RASA2 may modulate T cell proliferation, activation, and anti-tumor immunity.

    “RASA2, a GTPase-activating protein that stimulates the GTPase activity of wild-type RAS (Arafeh et al., 2015), has not been studied in the context of primary T cells and the immune system, but our findings suggest it may be a modulator of T cell proliferation, activation, and anti-tumor immunity.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    A GWAS of allergic diseases points to a credible missense risk variant in FAM105A.

    “Although little is known about FAM105A function, GWAS of allergic diseases implicates a credible missense risk variant in this gene (Ferreira et al., 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ferreira et al., 2017 source

      Not checked in the free run.

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

    Premisecitation

    CBLB is a known intracellular checkpoint; mouse studies show that targeting it in T cells improves tumor control.

    “Among these, CBLB has been best studied as an intracellular immune checkpoint that can be targeted in T cells to improve tumor control in mouse models (Chiang et al., 2007; Hinterleitner et al., 2012).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Chiang et al., 2007 source

      Not checked in the free run.

    • cites Hinterleitner et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    These genes are not general regulators of proliferation but modulate proliferation induced by TCR signaling.

    “Notably, targeting these genes did not increase proliferation in the unstimulated cells, indicating that they are not general regulators of proliferation but instead appear to modulate proliferation induced by TCR signaling.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The observed dose-response confirmed that most screen hits depend on the TCR stimulus and tune proliferative responses.

    “The observed dose-response confirmed that the majority of the screen hits are dependent on the TCR stimulus and serve to tune resulting proliferative responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    LCK directly phosphorylates and activates the TCR ITAMs and ZAP70.

    “gene targets that impaired TCR dependent proliferation included those encoding delta and zeta chains of the TCR complex itself (negative rank 18 and 6, respectively), and LCK (negative rank 20) which directly phosphorylates and activates the TCR ITAMs and the central signaling mediator ZAP70 (negative rank 299) (Dave et al., 1998; Tsuchihashi et al., 2000; Wang et al., 2010).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Dave et al., 1998 source

      Not checked in the free run.

    • cites Tsuchihashi et al., 2000 source

      Not checked in the free run.

    • cites Wang et al., 2010 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The SOCS1/TCEB2 complex restrains T cell responses and is a potential immunotherapy target.

    “Ablation of TCEB2, a binding partner of SOCS1, also improved tumor clearance, suggesting that the SOCS1/TCEB2 complex restrains T cell responses and is a potential target for immunotherapy (Ilangumaran et al., 2017; Kamizono et al., 2001; Liau et al., 2018).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Ilangumaran et al., 2017

      Not checked in the free run.

    • cites Kamizono et al., 2001 source

      Not checked in the free run.

    • cites Liau et al., 2018 source

      Not checked in the free run.

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

    Premisecitation

    Otulin (FAM105B) is a deubiquitinase essential for immune regulation.

    “A neighboring paralogue gene, Otulin (FAM105B), encodes a deubiquitinase with an essential role in immune regulation (Damgaard et al., 2016; Fiil and Gyrd-Hansen, 2016) (Figure S6D).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Damgaard et al., 2016 source

      Not checked in the free run.

    • cites Fiil and Gyrd-Hansen, 2016

      Not checked in the free run.

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

    Premisecitation

    RASA2 is a GTPase-activating protein that boosts GTP hydrolysis by wild-type RAS.

    “RASA2, a GTPase-activating protein that stimulates the GTPase activity of wild-type RAS (Arafeh et al., 2015), has not been studied in the context of primary T cells and the immune system, but our findings suggest it may be a modulator of T cell proliferation, activation, and anti-tumor immunity.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Arafeh et al., 2015 source

      Not checked in the free run.

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

    Study design and conductown data

    Normalized enrichment scores and p-values in the gene set enrichment analysis were determined by a permutation test with 10,000 iterations using same-size randomized gene sets and adjusted by the FDR method.

    “Normalized enrichment scores and p-values were determined by a permutation test with 10,000 iterations with same size randomized gene sets and adjusted with the FDR method.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Gene-level p-values in the screen analysis were obtained by a permutation test that randomizes guide assignments and were adjusted for false discovery using the Benjamini-Hochberg method.

    “The p-value for each gene is determined by a permutation test, randomizing guide assignments and adjusted for false discovery rates by the Benjamini–Hochberg method.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The proliferation index calculated from the fitted CFSE models is robust to variation in starting CFSE staining intensities.

    “This parameter is robust to variation in the starting CFSE staining intensities.”
    Quote in the paperNot a substantial claim

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

    Premisenone given

    Mouse studies have examined CBLB as an intracellular checkpoint whose targeting enhances CD8+ T cell tumor control.

    “Of these, CBLB has been studied in mouse models as an intracellular checkpoint that can be targeted to enhance tumor control by CD8+ T cells.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    SLICE allows more systematic searching for genetic perturbations that improve expansion and effector function in adoptive T cell therapies.

    “SLICE now provides an opportunity to search more systematically for genetic perturbations that enhance cell expansion and effector function for adoptive T cell therapies.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All donor replicates within each screen were grouped in the analysis to account for biological noise.

    “All donor replicates in each screen were grouped for analysis to account for biological noise.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In that patient, T cells carrying a lentiviral insertion in TET2 expanded preferentially at peak response and probably contributed to the complete response.

    “T cells with a lentiviral integration disrupting TET2 showed strong preferential expansion at the peak of a patient’s response and likely contributed to this complete response to CAR T cell therapy.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fraietta et al., 2018 source

      Not checked in the free run.

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

    Interpretationnone given

    Primary T cell screens can use various extracellular selective pressures or FACS-based phenotypic selections.

    “Primary T cell screens can be performed with various extracellular selective pressures and/or FACS-based phenotypic selections.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The fitted CFSE models were visually inspected and fitting parameters adjusted to minimize deviance from the original CFSE signal.

    “The fitted models were inspected visually, adjusting fitting parameters to minimize deviance from the original CFSE signal.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The gene set used as external reference for the in vivo immunotherapy hits in Figure 2E comprises 43 genes identified by Zhou et al. as having at least three shRNA guides enriched more than fourfold in tumor-tissue T cells relative to spleen.

    “For the external gene set for in vivo immunotherapy hits shown in Figure 2E, we used the 43 genes as determined by Zhou et al. (Zhou et al., 2014), having 3 or more shRNA guides with over 4-fold enrichment in T cells from tumor tissue compared to spleen.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zhou et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The authors developed a new pooled CRISPR screening technology that could explore unmapped genetic circuits in primary human cells and inform engineered cell therapy design.

    “In summary, we have developed a novel pooled CRISPR screening technology with the potential to explore unmapped genetic circuits in primary human cells and to guide the design of engineered cell therapies.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    In the pooled CRISPR screen analysis, non-targeting control guides were used to estimate the normalization size factor and to construct the mean-variance model for the null distribution used to call significant guide enrichment.

    “We used the non-targeting control guides to estimate the size factor for normalization, as well as to build the mean-variance model for null distribution, which is used to find significant guide enrichment.”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    All single-cell samples were processed simultaneously in the same experiment, and sample origin was not regressed out in the Seurat analysis.

    “Of note, all samples were processed simultaneously in the same experiment and thus sample origin was not regressed out (Butler et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Butler et al., 2018 source

      Not checked in the free run.

39 of 39

  • The paper relies on this without support.

    Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines.

    “Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines.”

  • The paper relies on this without support.

    Primary human T cells can only be cultured ex vivo for limited time spans.

    “primary human T cells – which can only be cultured ex vivo for limited time spans”
    “which are not immortalized and can only be expanded in culture for a limited amount of time”

  • The paper relies on this without support.

    Current pooled CRISPR screening methods rely on establishing cell lines with stably integrated Cas9 expression cassettes.

    “Current pooled CRISPR screening methods rely on establishing cell lines with stably integrated Cas9 expression cassettes.”

  • The paper relies on this without support.

    Systematic strategies to comprehensively analyze the function of genes that regulate human T cell responses are still lacking.

    “systematic strategies to comprehensively analyze the function of genes that regulate human T cell responses are still lacking”

  • The paper relies on this without support.

    For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.

    “Adoptive cell therapies that are effective for the treatment of solid tumors will require cells that can respond robustly to tumor antigens even in immunosuppressive tumor microenvironments.”

  • The paper relies on this without support.

    Proliferation in response to TCR stimulation is a broad phenotype governed by complex genetics.

    “We elected to use proliferation in response to TCR stimulation as our screening output, as it is a broad phenotype governed by complex genetics.”

  • The paper relies on this without support.

    CGS-21680 specifically targets ADORA2A.

    “ADORA2A – encoding the receptor specifically targeted by CGS-21680 – showed a high rank difference between the two treatment conditions (rank 19 in CGS-21680 vs rank 7399 in vehicle control), indicating that its knockout provided a specific escape from CGS-21680 (Figure 6A and Figure S6C).”

  • The paper relies on this without support.

    ADORA2B is not targeted by CGS-21680.

    “In contrast, ADORA2B, which is not targeted by CGS-21680, did not show a preferential proliferative advantage in the presence of this selective A2A agonist (Figure 6A).”

  • The paper relies on this without support.

    The parental CFSE signal peak can shift slightly from well to well in arrayed experiments.

    “As CFSE staining for arrays was done for individual populations of edited cells, the signal peak for the parental population might shift slightly from well to well.”

  • The paper relies on this without support.

    An unstimulated well is expected to have a single CFSE peak at the end of the assay.

    “for each well the stimulated well was compared to an identical unstimulated well, expected to have a single peak at the end of the assay.”

  • The authors state this as an assumption.

    CFSE profiles can be modeled as Gaussian distributions with log2-distanced peaks from successive cell divisions and CFSE dilution.

    “These CFSE profiles are modeled as Gaussian distributions, with log2 distanced peaks resulting from cell divisions and CFSE dilution.”
11 of 11

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?

  • Earlier studies limited differential expression testing to predefined candidate genes or grouped genes into signatures.

    “differential gene expression testing was restricted to pre-defined candidate genes7,9,10 or genes were grouped into gene signatures3,4,6,8.”

    From Targeted Perturb-seq enables genome-scale genetic screens in single cells (2020) · cites this paper as [3]

  • PROTACs have the potential to be first-in-class medicines in immuno-oncology as small-molecule drugs that target immune cell activation to phenocopy PD1/PDL1-directed agents.

    “PROTACs have the potential to be first-in-class medicines in immuno-oncology as small-molecule drugs that target immune cell activation to phenocopy PD1/PDL1-directed agents178,179.”

    From PROTAC targeted protein degraders: the past is prologue (2022) · cites this paper as [179]

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.

  • Genome-wide indexing PCR lists conflicting template volumes

    Minor

    The preparation method says 1 μL of purified product was taken for the second PCR, but its recipe specifies 5 μL of that product per reaction.

  • Pilot-screen CFSE staining has two different dates

    Minor

    The results place CFSE labeling 10 days after electroporation, whereas the screen methods place electroporation on Day 2 and CFSE staining on Day 14-12 days later.

2 of 2

Cites

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Related

Authors
Shifrut, E., Carnevale, J., Tobin, V., Roth, T. L., Woo, J. M., Bui, C. T., Li, P. J., Diolaiti, M. E., Ashworth, A., & Marson, A.
Affiliations
University of California, San Francisco (US)
Corresponding author
Marson, Alexander
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2018-12
Volume
175
Issue
7
Pages
1958-1971.e15
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2018.10.024
PMC
PMC6689405
OpenAlex
W2885096157
Cited by
573
References
84
Citations by year
2026: 79 · 2025: 74 · 2024: 76 · 2023: 83 · 2022: 80 · 2021: 80 · 2020: 65 · 2019: 32 · 2018: 2
FWCI
16.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • Parker Institute for Cancer Immunotherapy
  • Burroughs Wellcome Fund
  • Innovative Genomics Institute
  • NIH (P30 DK063720)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Oncology › CAR-T cell therapy research
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
primary human T cells, CRISPR screen, slices, T cell receptor signaling, T cell proliferation, cancer immunotherapy, adenosine signaling, immunosuppression, gene ablation, single-cell RNA sequencing
MeSH terms
CRISPR-Associated Protein 9, Humans, T-Lymphocytes, Genome, Human, Genome-Wide Association Study, Gene Knockout Techniques, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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