The DOI matches its Crossref record; 83 of the 84 references it cites were found in a registry or an index; none of the 32 sources its claims cite is checked for support yet.
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
Open accessPublished version of a preprint
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.
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56 Own result · 26 Own interpretation · 21 Not checked yet · 9 Own method · 1 No basis given
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
boosteddirect- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
boosteddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- 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.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
What the sources say
- Not checked yetcites Voisinne et al., 2016 source
hedgeddirect- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The results were well correlated between the primary and secondary screens (Figure 2C).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
boostedcorrelationaln = 4- Asserted: passes — The authors put it forward as true.
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None of the sources cited for this claim have been checked yet.
In the paper
“We found that sgRNAs targeting essential components of TCR signaling such as CD3D and LCP2, inhibited cell proliferation (de Saint Basile et al., 2004; Shen et al., 2009).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
What the sources say
plaindirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Top positive and negative regulators from the pilot screen were confirmed in both biological replicates of the GW screen along with numerous other hits (Figure 2A, B).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
plaincorrelationaln = 2- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
- 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.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our attempts to stably express Streptococcus pyogenes Cas9 by lentivirus in primary human T cells resulted in extremely poor transduction efficiencies.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
plain- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“sgRNAs targeting these genes were in the top 1% by rank difference in both biological replicates (Figure 1C).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
plaincorrelational- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In summary, several gene targets identified in the genome-wide screen for proliferative response to stimulation also potentiated in vitro tumor killing activity.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
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- It may state two separate claims as one. 29
- The quote only partly states this claim. 1
- Probably not a substantial claim. 15
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
boosteddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > Abstract
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- 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.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We conclude that sgRNA lentiviral infection with Cas9 protein electroporation (SLICE) results in effective and specific disruption of target genes.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- 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.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Paired with Single Cell RNA-Seq for Molecular Phenotyping of Modified Primary Human Cells
plaincorrelationaln = 2- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Paired with Single Cell RNA-Seq for Molecular Phenotyping of Modified Primary Human Cells
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
What the sources say
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Paired with Single Cell RNA-Seq for Molecular Phenotyping of Modified Primary Human Cells
plaincorrelational- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Arrayed Delivery of Cas9 RNPs Reveals that Hits Alter Stimulation Responses
plaindirectn = 4- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Arrayed Delivery of Cas9 RNPs Reveals that Hits Alter Stimulation Responses
plaincorrelationaln = 4- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Paired with Single Cell RNA-Seq for Molecular Phenotyping of Modified Primary Human Cells
hedgeddirectn = 2- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
plaindirectn = 4- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Arrayed Delivery of Cas9 RNPs Reveals that Hits Alter Stimulation Responses
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > Abstract
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
plaindirect- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Also stated in: § Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells · plain
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
hedgeddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one. The quote only partly states this claim.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
What the sources say
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
hedgedconditional- Asserted: passes — The authors put it forward as true.
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In the paper
“Although little is known about FAM105A function, GWAS of allergic diseases implicates a credible missense risk variant in this gene (Ferreira et al., 2017).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
What the sources say
- Not checked yetcites Ferreira et al., 2017 source
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
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- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
What the sources say
boosteddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Arrayed Delivery of Cas9 RNPs Reveals that Hits Alter Stimulation Responses
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
boosteddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Genome-wide Pooled CRISPR Screen Uncovers Regulators of the TCR Response
What the sources say
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
What the sources say
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
What the sources say
- Not checked yetcites Damgaard et al., 2016 source
- Not checked yetcites Fiil and Gyrd-Hansen, 2016
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > Screen Hits Boost Cancer Cell Killing in vitro by Engineered Human T Cells
What the sources say
- Not checked yetcites Arafeh et al., 2015 source
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“This parameter is robust to variation in the starting CFSE staining intensities.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
The paper states this without a source or data behind it here, so there is nothing to check it against. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“SLICE now provides an opportunity to search more systematically for genetic perturbations that enhance cell expansion and effector function for adoptive T cell therapies.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“All donor replicates in each screen were grouped for analysis to account for biological noise.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
What the sources say
- Not checked yetcites Fraietta et al., 2018 source
hedgedconditionalSingle case- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Primary T cell screens can be performed with various extracellular selective pressures and/or FACS-based phenotypic selections.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“The fitted models were inspected visually, adjusting fitting parameters to minimize deviance from the original CFSE signal.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
What the sources say
- Not checked yetcites Zhou et al., 2014 source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
hedged- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may state two separate claims as one.
In the paper
“Of note, all samples were processed simultaneously in the same experiment and thus sample origin was not regressed out (Butler et al., 2018).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
What the sources say
- Not checked yetcites Butler et al., 2018 source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
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.”
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?
In the citing paper
“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]
In the citing paper
“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.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > METHOD DETAILS
“Next 1 μL was taken from each purified PCR product to go into a second PCR for indexing.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > METHOD DETAILS
“Each reaction included 5μL of PCR product, 25μL of NEBNext 2x Master Mix (NEB, cat #M0541L), 1.25μL each of the 10μM p5-i5-read1 and read2-i7-p7 indexing primers, and water to 50μL total per reaction.”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > A Hybrid Approach to Introduce Traceable Genetic Perturbations into Primary Human T Cells
“At day 10 post-electroporation, cells were labeled with CFSE to track cell divisions and then TCR stimulated.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > STAR Methods > METHOD DETAILS
“24 hours after transduction (Day 2), cells were electroporated with Cas9 protein. Cells were then cultured in media with IL-2 at 50U/mL and split every two days, keeping a density of 1e6 cells/mL. On day 14, cells were CFSE stained and then restimulated”
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Metadata
- 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)
- 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
- bronze
- doi.org
- 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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