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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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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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
Strength:hedged· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesNone 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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“This validated the ability of many of the tested gene targets to increase T cell proliferation post stimulation, consistent with their robust effects in the pooled screens (Figure S3A).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Strikingly, we found that multiple guides targeting a previously uncharacterized gene, FAM105A (rank 15 in CGS-21680 vs. rank 13390 in vehicle control), were specifically enriched nearly to the same extent as ADORA2A (Figure 6B).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Targeting multiple negative regulators identified in the GW screens such as CD5, RASA2, SOCS1, and CBLB promoted the cluster 10–12 programs.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 noted that several guanine nucleotide binding proteins with potential roles in adenosine-responsive signaling had higher positive rank scores in the adenosine agonist GW screen, including GCGR (rank 35 vs. 1149), GNG3 (rank 199 vs. 12976), and GNAS (rank 836 vs. 2803).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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 screens identified dozens of genetic perturbations that positively and negatively modulate T cell proliferation.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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
“While many gene modifications promoted TCR proliferative responses to stimulation in the presence or absence of the adenosine receptor agonist, we identified several sgRNAs that were only enriched in the dividing cells in the presence of CGS-21680 (Figure 6A).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.
- Rests on an assumption: Proliferation in response to TCR stimulation is a broad phenotype governed by complex genetics.
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
“NY-ESO specific T cells started to cluster around RFP+ cancer cells at 12 hours, with more efficient cancer cell clearance at 36 hours for certain sgRNA targets compared to non-targeting controls (Figure 5B).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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, 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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passes- Rests on an assumption: ADORA2B is not targeted by CGS-21680.
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
“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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 found that T cells engineered to lack negative regulators of proliferation, such as SOCS1, CBLB, CD5, and others, also showed increased surface expression levels of both CD69 and CD154 compared to non-targeting control cells (Figure 3D and Figure S3C–D).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 similar gene targets appeared as positive and negative regulators across the conditions, the magnitude of the effects were blunted at higher levels of TCR stimulation, suggesting that stronger TCR stimulation can override the effects of these genetic perturbations (Figure 2D and Figure S2E).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Gene set enrichment analysis (GSEA) revealed significant overrepresentation of gene targets depleted from proliferating cells in the TCR signaling pathway (FDR < 0.01, Figure 2E and Figure S2F).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“A different shared activation program was observed as a result of targeting CD5, TCEB2, RASA2 or CDKN1B.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 programs tended to be characterized by the induction of markers of activation states (e.g. IL2RA, TNFRSF18/GITR), cell cycle genes (e.g. MKI67, UBE2S, CENPF and TOP2A), and effector molecules (e.g GZMB) (Figure 4F and Figure S4E).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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, guides against gene targets that were depleted in the proliferating cells in the pooled screens (CD3D and LCP2), showed a reduction in the proliferation index compared to the non-targeting controls.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“As expected, knockout of LCP2 – dentified in our screens as essential for a strong TCR stimulation response – robustly disabled T cell killing of A375 cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesNone of the sources cited for this claim have been checked yet.
In the paper
“Targeting SOCS1, a negative regulator of JAK/STAT signaling in T cells, showed enhanced T cell clearance comparable to CBLB (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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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, arrayed editing and phenotyping characterized the effects of genetic perturbations and revealed targets that boost stimulation-dependent proliferation and activation programs.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIn the paper
“Diacylglycerol (DAG) kinases, DGKA (rank 17) and DGKZ (rank 1), which are negative regulators of DAG-mediated signals, were both found to restrain human T cell proliferation following stimulation (Chen et al., 2016).”
§ 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
- Not checked yetcites Chen et al., 2016 source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIn the paper
“Many of the negative regulators in our screen were enriched in the list of hits from a published shRNA screen (Zhou et al., 2014) that was designed to discover gene targets that boost T cell proliferation in tumor tissue in vivo (FDR < 0.01).”
§ 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
- Not checked yetcites Zhou et al., 2014 source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Knockout of CBLB tended to induce a cell state signature similar to targeting known checkpoint genes BTLA or LAG3, as evidenced by similarity in cluster representation (Figure S4F).”
§ 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
Strength:hedged· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
“Targets depleted from proliferating cells in this GW screen encode key protein complexes critical for TCR signaling (Figure 2F).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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, stimulated cells generally induced expression of cell cycle genes (e.g.: MKI67) and cytolytic granzymes (e.g.: GZMB) (Figure 4B).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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, unstimulated cells largely expressed markers of a resting state, such as 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
Strength:plain· not yet calibratedn = 2Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
“Synthetic bulk gene expression profiles showed that stimulated cells up-regulated many cell cycle genes, indicating response to TCR stimulus (Figure S4B).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
“Conversely, targeting a positive regulator of TCR signaling, LCP2, reduced expression of CD69 and CD154 in stimulated cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Importantly, 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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: CGS-21680 specifically targets ADORA2A.
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 found that targeting each of these genes with two different sgRNAs each led to resistance to suppression by CGS-21680, as predicted by our screen (Figure 6C).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.
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
“Stimulated cells were enriched in clusters 9–12, which were characterized by preferential expression of mitotic cell cycle and T cell activation cellular programs (Figure S4C).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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
“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
Load-bearingStrength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Importantly, these edits did not lead to increased T cell proliferation in the absence of TCR stimulation, suggesting that they selectively overcome CGS-21680 suppression of TCR stimulation.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 demonstrated that a suppressive pressure can be added to the screen, in our case an adenosine agonist, to identify gene perturbations that confer resistance.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Lastly, we showed that ADORA2A and FAM105A targeted T cells were resistant to suppression by CGS-21680 in the in vitro cancer cell killing assay (Figure S6E).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 focused on CD8+ T cells, but showed that SLICE also can be employed in CD4+ T cells, and eventually may be generalizable to many other primary human cell types.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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 found that ablation of at least four targets (SOCS1, TCEB2, RASA2, and CBLB) in human T cells enhanced both proliferation and in vitro anti-cancer function.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesNone of the sources cited for this claim have been checked yet.
In the paper
“We previously showed efficient gene editing of primary human T cells by electroporation of Cas9 protein pre-loaded in vitro with sgRNAs (Hultquist et al., 2016; Schumann et al., 2015).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesWhat 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
“Integrated analysis of the two independent screens performed on a total of four human blood donors provided improved power for target discovery, particularly for negative regulators of T cell proliferation (Table S4 and Figure S2D).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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 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
“This low efficiency was prohibitive of large-scale pooled screens in primary cells, which are not immortalized and can only be expanded in culture for a limited amount of time.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: Primary human T cells can only be cultured ex vivo for limited time spans.
In the paper
“Targeting SOCS1, a negative regulator of JAK/STAT signaling in T cells, showed enhanced T cell clearance comparable to CBLB (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
- Not checked yetcites Liau et al., 2018 source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesWhat 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
“Efficient editing for the majority of gene targets was validated by reduced expression of sgRNA target transcripts compared to levels in cells with non-targeting control sgRNAs (Figure S4D).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“This arrayed RNP gene targeting system demonstrated that the majority of top genes identified by our screens robustly modulate stimulation-dependent proliferation in human CD8+ T cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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 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
“These data also demonstrate that targeting negative regulators of proliferation can induce specialized stimulation-dependent effector gene programs that could enhance the potency of T cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“These results suggest an important role for FAM105A in mediating adenosine immunosuppressive signals in T cells.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.
In the paper
“The E3 ubiquitin-protein ligase, CBLB (rank 4) and its interacting partner, CD5 (rank 12), work together to inhibit TCR activation via ubiquitination leading to degradation of the TCR (Voisinne et al., 2016).”
§ 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
- Not checked yetcites Voisinne et al., 2016 source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA 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 integration of SLICE pooled CRISPR screens and single cell RNA-Seq provides a powerful approach to both discover and characterize critical gene pathways in primary human cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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 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
“Importantly, 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).”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: CGS-21680 specifically targets ADORA2A.
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
“Because these CRISPR-based target perturbations require pre-stimulation, our results are generally expected to reflect modulators of the TCR-response in antigen-experienced cells.”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesNone of the sources cited for this claim have been checked yet.
In the paper
“UBASH3A (rank 10), TNFAIP3, and its partner TNIP1 (rank 13 and 24, respectively) inhibit TCR-induced NFkB signaling, a critical survival and growth signal for activated CD8+ T cells (Düwel et al., 2009; Ge et al., 2017).”
§ 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
- Not checked yetcites Düwel et al., 2009 source
- Not checked yetcites Ge et al., 2017
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- 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 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
“These gene targets appear to play a selective role in adenosine receptor-mediated T cell suppression.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.
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
“While similar gene targets appeared as positive and negative regulators across the conditions, the magnitude of the effects were blunted at higher levels of TCR stimulation, suggesting that stronger TCR stimulation can override the effects of these genetic perturbations (Figure 2D and Figure S2E).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Importantly, these edits did not lead to increased T cell proliferation in the absence of TCR stimulation, suggesting that they selectively overcome CGS-21680 suppression of TCR stimulation.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Thus, we identified both extracellular and intracellular targets that could be modified to generate T cells resistant to adenosine suppression.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“However, the reproducible trends of top targets across multiple donors and screens, as well as in the RNP validation experiments, suggest that consistent hits are most likely not determined by heterogeneity of the starting populations.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Load-bearingStrength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Integration of these CRISPR-based functional genetic studies rapidly pinpointed genes in human T cells that can be targeted to enhance stimulation-dependent proliferation, activation responses, effector programs, and in vitro cancer cell killing.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIn the paper
“The potential power of human primary T cell loss-of-function screens was recently demonstrated in a patient who received CAR T cell therapy for chronic lymphocytic leukemia (CLL) (Fraietta et al., 2018).”
§ 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 screens can be performed routinely at large-scale in primary cells from multiple human donors, ensuring biologically reproducible discoveries.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 SLICE pooled screening approach is flexible and versatile as it can be adapted to probe diverse genetic programs that regulate primary T cell biology.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“This exemplifies the potential for this platform to be used to discover gene targets that can enhance specialized T cell functions.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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 provides a new platform for genome-wide CRISPR loss-of-function screens in primary human T cells, a cell type that has been central to a revolution in cancer immunotherapies.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA 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
“Overall, these findings demonstrate that SLICE is able to identify both known and novel components of a pathway required for primary cell-response to a specific extracellular cue.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > RESULTS > SLICE Screen for Resistance to Immunosuppressive Adenosine Signaling
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: For solid tumors, effective adoptive cell therapies need T cells that can respond strongly to tumor antigens despite immunosuppressive microenvironments.
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
“Our data suggest that RASA2 and the SOCS1/TCEB2 complex members may also be targets for modulation in immunotherapies.”
§ Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function > DISCUSSION
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
- 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
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
Strength:hedged· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Strength:boosted· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably 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
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably 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
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably 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
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably 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
Load-bearingStrength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt 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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably 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
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably 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.
Genome-wide indexing PCR lists conflicting template volumes
MinorThe 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.
“Next 1 μL was taken from each purified PCR product to go into a second PCR for indexing.”
“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.”
Pilot-screen CFSE staining has two different dates
MinorThe 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.
“At day 10 post-electroporation, cells were labeled with CFSE to track cell divisions and then TCR stimulated.”
“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”
Citations
Cites
Integrating single-cell transcriptomic data across different conditions, technologies, and species
Butler, A., Hoffman, P., Smibert, P., Papalexi, E., & Satija, R. (2018) · Nature Biotechnology · doi:10.1038/nbt.4096 · Cited by 13,174
Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer
Reck, M., Rodríguez-Abreu, D., Robinson, A. G., Hui, R., Csőszi, T., Fülöp, A., Gottfried, M., Peled, N., Tafreshi, A., Cuffe, S., et al. (2016) · New England Journal of Medicine · doi:10.1056/nejmoa1606774 · Cited by 10,089
UMAP: Uniform Manifold Approximation and Projection
McInnes, L., Healy, J., Saul, N., & Großberger, L. (2018) · Journal of Open Source Software · doi:10.21105/joss.00861 · Cited by 9,826
UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction
McInnes, L., Healy, J., & Melville, J. (2018) · arXiv · doi:10.48550/arxiv.1802.03426 · Cited by 7,101
The Reactome pathway Knowledgebase
Fabregat, A., Sidiropoulos, K., Garapati, P., Gillespie, M., Hausmann, K., Haw, R., Jassal, B., Jupe, S., Korninger, F., McKay, S., et al. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkv1351 · Cited by 5,605
Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy
Sharma, P., Hu-Lieskovan, S., Wargo, J. A., & Ribas, A. (2017) · Cell · doi:10.1016/j.cell.2017.01.017 · Cited by 5,588
Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma
Wolchok, J. D., Chiarion-Sileni, V., Gonzalez, R., Rutkowski, P., Grob, J.-J., Cowey, C. L., Lao, C. D., Wagstaff, J., Schadendorf, D., Ferrucci, P. F., et al. (2017) · New England Journal of Medicine · doi:10.1056/nejmoa1709684 · Cited by 5,357
Improved vectors and genome-wide libraries for CRISPR screening
Sanjana, N. E., Shalem, O., & Zhang, F. (2014) · Nature Methods · doi:10.1038/nmeth.3047 · Cited by 5,239
Genome-scale CRISPR-Cas9 knockout screening in human cells
Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G., et al. (2014) · Science · doi:10.1126/science.1247005 · Cited by 5,234
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
Flagged first, then by citations
1 of the 84 references couldn’t be matched to a known paper.
Cited by
Sorted by citations
PROTAC targeted protein degraders: the past is prologue
Békés, M., Langley, D. R., & Crews, C. M. (2022) · Nature Reviews Drug Discovery · doi:10.1038/s41573-021-00371-6 · Cited by 3,395
Recent advances and discoveries in the mechanisms and functions of CAR T cells
Larson, R. C., & Maus, M. V. (2021) · Nature Reviews Cancer · doi:10.1038/s41568-020-00323-z · Cited by 852
Natural killer cells in antitumour adoptive cell immunotherapy
Laskowski, T. J., Biederstädt, A., & Rezvani, K. (2022) · Nature reviews. Cancer · doi:10.1038/s41568-022-00491-0 · Cited by 810
Treg cell-based therapies: challenges and perspectives
Raffin, C., Vo, L. T., & Bluestone, J. A. (2019) · Nature reviews. Immunology · doi:10.1038/s41577-019-0232-6 · Cited by 746
CAR immune cells: design principles, resistance and the next generation
Labanieh, L., & Mackall, C. L. (2023) · Nature · doi:10.1038/s41586-023-05707-3 · Cited by 695
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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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