The DOI matches its Crossref record; 75 of the 77 references it cites were found in a registry or an index; 4 of the 46 sources its claims cite were checked for support.
Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
Open accessAbstract
SUMMARY Adoptive transfer of genetically-modified immune cells holds great promise for cancer immunotherapy. CRISPR knock-in targeting can improve cell therapies, but more high-throughput methods are needed to test which knock-in gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates, and applied it to perform pooled knock-in screens in primary human T cells. Pooled knock-in of dozens of unique barcoded templates into the TCR-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed Pooled Knock-In Sequencing (PoKI-Seq), combining single-cell transcriptome analysis and pooled knock-in screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel TGFβR2–41BB chimeric receptor that improved solid tumor clearance. Pooled knock-in screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knock-in programs for cell therapies.
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Claims the paper makes
58 Own result · 39 Own interpretation · 22 Not checked yet · 8 Own method · 6 No basis given · 2 Supported
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In the paper
“The pooled knock-in screens confirmed the functional effects of a recently described truncated Fas receptor (Yamamoto et al., 2019) included as a positive control”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
What the sources say
- Supportedcites Yamamoto et al., 2019 source
The cited paper describes a truncated Fas receptor, Fas ΔDD, and reports that T cells expressing it were protected from FasL-induced apoptosis (Figure 7 and accompanying results). The pooled knock-in screens are the citing paper’s own finding; this citation identifies the previously described receptor and its functional effect. (LLM, conf=0.91)
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- Supported
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In the paper
“We first demonstrated that expected DNA barcodes could be selectively sequenced from cells expressing the corresponding knock-in encoded protein products.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“Sorting cells with successful HDR knock in (based on expression of the NY-ESO-1 TCR) that co-expressed either GFP+ or RFP+ strongly enriched for the appropriate DNA barcode isolated sequenced from either gDNA or cDNA (Figure 1C and Figure S2C–E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“However, as expected, primers complementary to the template homology arm mismatches – which should preferentially amplify non-HDR integrated templates – did not show the same enrichment for correct barcodes in the sorted populations (Figure 2E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“Template switching varied based on the stage of library production when constructs were pooled, and we chose to pool all subsequent knock-in construct libraries at the electroporation stage (Figure 1E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“Finally, we were able to readily track the relative abundances of knock-in construct barcodes over time in an expanding population of T cells cultured in interleukin 2 (IL-2).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“This platform nominated a novel TGFβR2–41BB chimeric receptor that improved solid tumor clearance.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > Abstract
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In the paper
“We next directly validated the homology arm mismatch sequencing strategy (Figure 1B) to selectively amplify on-target barcodes using the larger 36-member pooled knock-in library.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“PCR primers that anneal to the inserted knock-in sequence and to the genomic flanking sequence can then selectively amplify successful on-target HDR events (Figure S2A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“Following anti-CD3/CD28 stimulation, we observed a striking enrichment of constructs encoding various synthetic versions of the Fas receptor (Figure 3B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The in vitro conditions used in the pooled screens mimic aspects of tumor microenvironments.
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In the paper
“Only ~25% of cells showed evidence of multiple integrations (Figure 1D and Figure S2E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“and a short DNA sequence that does not match the target genomic locus can be introduced into one homology arm without a large reduction in knock-in efficiency (Figure 1B and Figure S2B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates, and applied it to perform pooled knock-in screens in primary human T cells.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > Abstract
plain- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: The field lacks rapid methods to test and directly compare the functions of large DNA knock-in integrations in human primary cells.
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
“As expected, the percentage of reads with the GFP or RFP sequencing barcodes closely corresponded to the percentage of GFP or RFP positive cells observed at the protein level across four human donors (Figure 2D).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
plaincorrelationaln = 4- Asserted: passes — The authors put it forward as true.
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In the paper
“Addition of a barcode unique for each insert within degenerate bases of the TCR-α sequence enabled a rapid DNA sequence readout by PCR of the abundance of each individual on-target insert in the pooled population by either of two complementary methods: 1) sequencing directly from genomic DNA (gDNA), or 2) sequencing from reverse transcribed mRNA (cDNA) (Figure 1B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“For both gDNA and cDNA sequencing conditions, when GFP+ or RFP+ cells were sorted from the cells with successful on-target knock-in (NY-ESO-1 TCR+), the correct barcodes were selectively sequenced when using primers that bound the genomic sequence at the rate predicted when taking multiple integrations and template switching into account (Figure 2E and STAR Methods).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“With this pooling strategy only ~10% of cells showed evidence of template switching (e.g. nine in ten knock-in positive cells will have the correct combination of knock-in construct and barcode) (Figure 1E and Figure S2C–J).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“We developed a deep sequencing strategy that selectively captures on-target knock-in constructs while avoiding NHEJ-edited or un-edited target genomic sequences, non-integrated HDR templates, or off-target integrations (Figure 1B and Figure S2A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“Quantitative barcode sequencing demonstrated that even with the larger library all knock-in constructs were well-represented across multiple pooled knock-in experiments performed in four independent human donors (Figure 2A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“Pooled knock-in of dozens of unique barcoded templates into the TCR-locus revealed gene constructs that enhanced fitness in vitro and in vivo.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > Abstract
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In the paper
“We further developed Pooled Knock-In Sequencing (PoKI-Seq), combining single-cell transcriptome analysis and pooled knock-in screening to measure cell abundance and cell state ex vivo and in vivo.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > Abstract
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- Rests on an assumption: Cell abundance alone is insufficient to assess cellular function, and an ideal screening method should also assess modified cell states.
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In the paper
“~ 90% of barcodes from sorted GFP+ cells were correct (Figure S2J).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Quantitative barcode sequencing demonstrated that even with the larger library all knock-in constructs were well-represented across multiple pooled knock-in experiments performed in four independent human donors (Figure 2A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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In the paper
“In contrast, a truncated CTLA4 construct showed reduced cell killing (Figure 4D–E), consistent with its impaired fitness in the pooled knock-in screens (although diminished proliferation assessed by CFSE was not observed with this individual construct, Figure S5B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
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In the paper
“Sorting GFP+ or RFP+ cells from the pooled knock-in positive population (NY-ESO-1 TCR+) strongly enriched for the expected template barcodes, confirming the ability of PoKI-Seq to accurately assign specific knock-in constructs to cells (Figure 5B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“For example, in the presence of exogenous TGFβ, TGFβR2-derived chimeric receptors promoted continued expression of various hallmark proliferative genes such as MKI67 and TOP2A, while these genes were inhibited by TGFβ in cells expressing other KI constructs.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Distinct clusters of cell states emerged, especially with addition of exogenous TGFβ (Figure 5D–E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Density plots for individual knock-in constructs revealed significant cell state differences in T cells modified with TGFβR2-derived constructs compared to control and non-TGFBR2-derived constructs in the stimulation plus TGFβ culture condition (Figure 5G).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Although this was not the phenotype initially tested in the pooled screens, the TGFβR2–41BB increased target in vitro cancer cell killing in T cells from four human blood donors across a range of effector to target cell ratios (Figure 4D–E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
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In the paper
“In contrast, a truncated CTLA4 protein decreased T cell fitness in the same conditions across four donors”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
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In the paper
“also identified novel synthetic Fas switch receptors, including one with a 4–1BB intracellular domain, that also promoted T cell fitness in stimulated conditions (Figure 3B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“In the presence of exogenous TGFβ, the TGFβR2–41BB construct successfully rescued the impaired cancer cell killing across experiments performed from four healthy human donors (Figure 4F).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
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In the paper
“The overall proportion of cells assigned two knock-in constructs (Figure 5C) closely corresponded with predicted frequencies based on GFP/RFP 2-member library experiments (Figure 1D).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Finally, addition of the immunosuppressive cytokine TGFβ1 gave cells expressing engineered TGFβR receptors a selective fitness advantage, including both a previously described truncated TGFβR2 receptor (Gorelik and Flavell, 2000; Kim et al., 2017; Kloss et al., 2018) included as a positive control and a novel chimeric TGFβR2–41BB receptor (Figure 3D and Figure S4D–E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
What the sources say
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Specifically, cells possessing TGFβR2-derived constructs were more likely to be found in clusters otherwise associated with the stimulation-only condition rather than the stimulation + TGFβ condition, including cluster 8, characterized by genes associated with cell proliferation, and cluster 12, characterized by genes associated with cell killing (Figures 5E, H and S6E–F).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Due to the lower number of human T cells isolatable from individual solid tumor xenografts (generally 10,000–20,000 cells per mouse), greater variance between replicates and donors was observed in the in vivo screens than in the in vitro screens (Figure S7B–C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
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In the paper
“Single cell template barcode assignments were confirmed further as transcripts encoded by corresponding knock-in constructs tended to be expressed at increased levels, similar to what had been observed at the protein level with delivery of individual knock-in constructs (Figure 4B and Figure S6D).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Consistent with TCF7 promoting TIL abundance, but not promoting mature effector cytokine expressing cells, the TCF7 cell population did not enhance tumor control relative to tNGFR controls (Figure 7B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > A novel TGFβR2–41BB chimeric receptor nominated by in vitro and in vivo pooled knock-in screens improves solid tumor clearance by primary human T cells
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In the paper
“As expected, when sorted GFP or RFP positive cells were assigned two knock-in constructs, at least one of the knock-in constructs assigned was almost always either GFP or RFP respectively (Figure 5C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“Each knock-in construct caused distinct enrichment patterns in individual cell clusters in both TCR stimulation and stimulation plus TGFβ conditions that broadly corresponded to results from the in vitro pooled knock-in screens (Figure S6E).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Similarly, the TGFβR2-derived knock-in constructs were relatively depleted from cells in the clusters otherwise promoted by TGFβ treatment (Clusters 2, 4 and 6) (Figure 5H).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“In contrast, the TGFβR2–41BB receptor construct that promoted abundance and effector cytokine gene expression in TILs markedly reduced tumor sizes (P < 0.0001 at day 33 vs TCR+tNGFR).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > A novel TGFβR2–41BB chimeric receptor nominated by in vitro and in vivo pooled knock-in screens improves solid tumor clearance by primary human T cells
plaindirectn = 4 ·- Asserted: passes — The authors put it forward as true.
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In the paper
“The TCF7 knock-in construct that only showed mild effects in vitro (not reaching statistical significance) promoted accumulation of T cells in the tumor microenviroment across four donors (Figure 6B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
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In the paper
“TCR stimulation without anti-CD28 co-stimulation selected for various chimeric receptors with intracellular CD28 domains such as TIM3-CD28 and CTLA4-CD28 (Figure S4C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
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In the paper
“Hierarchical clustering revealed that exposure to TGFβ drove the greatest transcriptional changes (Figure 5I and Figure S6G).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
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In the paper
“This lead construct indeed showed markedly enhanced clearance of an in vivo solid tumor model.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
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In the paper
“Increased cell expansion induced by the TGFβR2–41BB construct correlated with evidence of increased cell proliferation in the presence of TGFβ (measured by CFSE dilution relative to control cells) (Figure 4C and Figure S5B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
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In the paper
“the rate of template switching in the pooled electroporation condition used throughout the study was determined to be ~10% (Figure 1E)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
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In the paper
“Biallelic knock-in rate was estimated to be ~25% of total knock-in positive cells (Figure 1D and Figure S2E)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
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In the paper
“These results showed that engineered cell phenotypes could be quantitatively tracked in pooled knock-in experiments through DNA barcode sequencing.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
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In the paper
“Pooled screening approaches have been challenged by template switching that complicate interpretation of DNA barcode readouts (Hanna and Doench, 2018; Hegde et al., 2018; Sack et al., 2016).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
What the sources say
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In the paper
“Briefly, the majority of a HDR template’s homology arm sequences are not incorporated with on-target HDR integrations (Paquet et al., 2016)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
What the sources say
- Not checked yetcites Paquet et al., 2016 source
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“More recent work has revealed potential advantages of targeted integrations of antigen receptor genes into defined genomic loci (Eyquem et al., 2017), which can be achieved with programmable nucleases such as CRISPR-Cas9 along with viral or non-viral knock-in templates (Gwiazda et al., 2016; Eyquem et al., 2017; Roth et al., 2018).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
- Not checked yetcites Eyquem et al., 2017 source
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In the paper
“In addition, multiple barcoded templates introduced in the same cell could complicate interpretation of each one’s effects (Feldman et al., 2018; Roth et al., 2018).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled non-viral knock-in targeting of barcoded DNA templates
What the sources say
- Not checked yetcites Feldman et al., 2018
- Not checked yetcites Roth et al., 2018 source
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In the paper
“The excessive antigen density and kinetics of antigen exposure in solid tumors can drive exhaustion or dysfunction of T cells (Khan et al., 2019; Wherry, 2011).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
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In the paper
“Suppressive cytokines such as TGFβ can be co-opted by tumors to inhibit T cell function (Dahmani and Delisle, 2018; Thomas and Massagué, 2005).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
- Not checked yetcites Dahmani and Delisle, 2018
- Not checked yetcites Thomas and Massagué, 2005 source
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Tregs, myeloid derived suppressor cells (MDSCs), and dendritic and stromal cell populations within the tumor can deprive T cells of necessary co-stimulatory signals or induce inhibitory signals (Chen and Mellman, 2013; Ostrand-Rosenberg et al., 2012).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Next-generation cellular immunotherapies will likely need to be engineered with enhanced function to overcome these challenges presented by solid tumors (Rafiq et al., 2019).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
- Not checked yetcites Rafiq et al., 2019
hedgedconditional- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: CAR T cells have not proved effective for solid tumors.
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 gene ablation can enhance cell therapies, the translational potential for knock-in of large gene cassettes to re-write cell programs is even greater.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
plain- 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
“Coupling pooled CRISPR gene knockouts with single cell RNA sequencing provides a powerful approach to assess the effect of each gene perturbation on cell state (Adamson et al., 2016; Datlinger et al., 2017; Dixit et al., 2016; Jaitin et al., 2016).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
boostedcorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“Pooled knock-in screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knock-in programs for cell therapies.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > Abstract
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In the paper
“Barcoded pools of non-viral HDR templates can now be used to explore the in vitro and in vivo functions of diverse sequence variants at targeted coding and non-coding elements in the genomes of primary human cells.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
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In the paper
“Taken together, pooled knock-in screening combined with scRNA-seq can rapidly reveal new DNA sequences with potential to enhance therapeutic T cell functions.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > A novel TGFβR2–41BB chimeric receptor nominated by in vitro and in vivo pooled knock-in screens improves solid tumor clearance by primary human T cells
plaindirectn = 4 ·- Asserted: passes — The authors put it forward as true.
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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
“Effective barcoding of targeted knock-in sequences also allowed single-cell transcriptome readouts in a pool of modified cells to assess the effects of each knock-in construct on cell state, a method we term Pooled Knock-In Sequencing (PoKI-seq).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“These data from pooled knock-in screens highlight condition-specific functional enhancements conferred by synthetic proteins or forced expression of key regulators.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
plaindirect- Asserted: passes — The authors put it forward as true.
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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
“Targeted non-viral pooled knock-in screens have notable advantages over other systems that potentially could be used to discover effects of gene gain-of-function.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: Technical challenges have limited CRISPRa applications in therapeutically relevant primary human cell types.
- Rests on an assumption: CRISPRa can only induce natural gene products encoded in the genome and does so with variable efficiency.
- Rests on an assumption: A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.
- Rests on an assumption: Most over-expression studies have been performed with lentiviral or retroviral vectors.
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
“Although the pooled screens focused on cell-intrinsic effects on T cell fitness, they also successfully identified novel gene constructs that can enhance in vitro anti-cancer cell efficacy.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
plaindirectn = 4- Asserted: passes — The authors put it forward as true.
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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
“Here we overcame these technical limitations with a widely adaptable technology to barcode and track on-target integrations of HDR templates in a pooled library.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
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- Rests on an assumption: A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.
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 results of these pooled targeted knock-in screens were reproducible across multiple biological replicates, technical replicates, sorting conditions and pooling conditions (Figure S4F–H).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
plain- Asserted: passes — The authors put it forward as true.
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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
“Each knock-in construct can be tracked in the targeted cell population under diverse in vitro and in vivo conditions to reveal the most effective modifications.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“but TCF7 expression was lost upon differentiation into effector cells (Siddiqui et al., 2019).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
What the sources say
- Not checked yetcites Siddiqui et al., 2019 source
plaindirect- Asserted: passes — The authors put it forward as true.
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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
“More generally PoKI-Seq was able to reveal target gene pathways and context-dependent changes in cell state modulated by individual knock-in constructs.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plaindirect- Asserted: passes — The authors put it forward as true.
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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 pooled knock-in platform presented here offers a new opportunity to screen systematically for large gene constructs that can be added to a T cell to modulate critical functions for effective cell therapies.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plain- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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- Rests on an assumption: The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.
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
“Overall, these experiments confirmed the molecular biology of PoKI-seq and also demonstrated its ability to phenotype individual cells with either single or multiple knock-in constructs.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
boosted- Asserted: passes — The authors put it forward as true.
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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
“Non-viral pooled knock-in screening will accelerate the discovery and development of synthetic DNA sequences to reprogram the specificity and function of adoptive cellular therapies.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: unclear — It is unclear whether it could 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: The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.
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 findings validated the pooled library-based approach to identify individual knock-in sequences to engineer T cell function.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
plaindirectn = 8- 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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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
“PoKI-seq offers a complementary single-cell approach to study large knock-in perturbations in pooled experiments.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plain- Asserted: passes — The authors put it forward as true.
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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
“Knock-in of large genetic constructs enables the engineering of synthetic receptors to re-wire the signal T cells receive when exposed to TGFβ in the tumor microenvironment, including switching the suppressive TGFβ signal to a stimulatory 41BB signal.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
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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
“PoKI-Seq confirmed the shared biological effects of multiple TGFβR2-derived receptors, which convert a suppressive signal into a signal that promotes cell proliferation and an effector cell state.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plaindirect- Asserted: passes — The authors put it forward as true.
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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 sources of variability could complicate interpretation of pooled screens, introducing both false positives and false negatives.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
hedgedconditional- Asserted: passes — The authors put it forward as true.
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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
“likely due to competition for CD28 co-stimulatory signaling (Figure 3B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
hedgedconditionaln = 4- 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.
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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 preclinical success of a novel construct nominated by pooled knock-in screens highlights the power of the platform.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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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 results underscore the power of complementary in vitro and in vivo assays to assess synthetic gene functions.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
boostedn = 4- 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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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 metrics together established the fidelity of barcode assignment to single cell transcriptomes for the PoKI-Seq platform.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plaindirectn = 2- 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.
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
“Simplifying the equation yields: 3)Y/X=2+2*X+2*X2+2*X3+…+2*Xn 4)Y/X=2+Y 5)(Y/X)−Y=2 6)Y*(1/X−1)=2 7)Y=2/(1/X−1)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plaincorrelational- Asserted: passes — The authors put it forward as true.
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- The authors state this as an assumption: Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.
- The authors state this as an assumption: A template that has switched once is equally likely to switch again.
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
“Therefore, out of 1250 total alleles, 1000 would be predicted to contain the correct knock-in construct/barcode before accounting for template switching.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
hedged- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- The authors state this as an assumption: The knock-in library is arbitrarily large.
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 a library of arbitrarily large size, there will be no significant switching with templates containing the same barcode, and thus the observed and actual amounts of template switching will converge.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
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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 total then, 900 out of 1250 alleles, or 72% of alleles, will contain the correct knock-in barcode.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
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
“Half of the alleles in biallelic knock-in cells will have a different knock-in construct/barcode than the sorted (GFP) knock-in construct/barcode, assuming an arbitrarily large knock-in library.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- The authors state this as an assumption: The knock-in library is arbitrarily large.
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
“Assuming a 10% rate of template switching, 100 out of 1000 alleles with the correct knock-in construct (GFP) will contain a barcode for a different construct.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
plain- Asserted: passes — The authors put it forward as true.
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- 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. 36
- Probably not a substantial claim. 13
Every source the paper cites for this claim was checked and supports it.
In the paper
“Genome-scale pooled CRISPR loss-of-function screens now allow forward genetic studies in many mammalian cell types (Wang et al., 2014; Shalem et al., 2014) including primary T cells where they have identified genes that may be targeted in future immunotherapies (Dong et al., 2019; Shifrut et al., 2018; Wei et al., 2019).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
- Supportedcites Wang et al., 2014 source
Wang et al. is cited for the first clause, not the subsequent claim about primary T cells and immunotherapies, which has separate citations. The Abstract describes a genome-scale pooled CRISPR loss-of-function screen in two human cell lines and concludes that it enables systematic genetic analysis in mammalian cells. (LLM, conf=0.90)
- Supportedcites Shalem et al., 2014 source
The Abstract (page not available in our copy) reports pooled genome-scale CRISPR-Cas9 knockout screens in human cancer and pluripotent stem cells, supporting the claim attached to Shalem et al. The primary-T-cell and immunotherapy findings are attached to different citations in the citing sentence, not to this paper. (LLM, conf=0.90)
- Supportedcites Dong et al., 2019 source
The Results section describes a genome-scale sgRNA library used for CRISPR screens in primary CD8 T cells and reports genes identified as potential cancer immunotherapy targets. The broader claim about screens across many mammalian cell types is attached to other citations in the citing sentence, not to Dong et al. (LLM, conf=0.93)
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.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Supported
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 library contained large knock-in constructs ranging from 2 kb to 3 kb and even the largest constructs were well-represented across these biological replicates (Figure 2B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
plainn = 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
“Barcode abundance was maintained throughout expansion for all constructs, with a notable exception of the knock-in construct encoding IL2RA, the high affinity receptor for the interleukin (IL-2), which enriched over time in culture consistent with its known roles promoting T cell fitness (Figure 2F–G).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
plain- 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
“the TGFβR2–41BB knock-in induced a distinct transcriptional program compared to TCF7 knock-in cells, with higher expression of effector cytokines such as IFN-ɣ that could enhance tumor clearance.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
plaindirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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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
“While both TCF7 and TGFβR2–41BB promoted expression of genes such as TNFSFR9 (4–1BB) relative to control cells”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
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.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
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
“Barcodes could be sequenced from either cDNA or genomic DNA”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
hedged- Asserted: passes — The authors put it forward as true.
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- 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 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
“With excessive stimulation, which partially mirrors antigenic abundance in tumor environments, the transcription factor TCF7 (encoding the protein TCF1) slightly increased cell abundance, although this effect did not reach statistical significance (Figure S4B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
plain- 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.
- 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
“Cells with the TGFβR2–41BB construct were the only ones that successfully cleared the solid tumor model at the doses tested here, an effect that was reproducible in many of the mice tested with modified T cells from 4 human blood donors (Figure 7B–C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > A novel TGFβR2–41BB chimeric receptor nominated by in vitro and in vivo pooled knock-in screens improves solid tumor clearance by primary human T cells
plaindirectn = 4 ·- 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
“Indeed, the anti-apoptotic FAS-41BB chimera and anti-suppressive TGFβR2–41BB chimera both promoted context-dependent increased expansion relative to controls (Figure 4C and Figure S5B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
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
“Clustering of knock-in constructs across all genes differentially expressed in the identified single cell clusters showed strong similarity between the TGFβR2-derived constructs in the presence of TGFβ and revealed downstream target genes that are modulated by the receptors.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
boostedcorrelational- 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
“Strikingly, the TCF7 construct was relatively enriched in CCR7+ TILs, while TGFβR2–41BB was relatively enriched in both IL-2+ and IFNG+ TIL populations (Figure 6D).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
plaincorrelationaln = 2- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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It may state two separate claims as one.
In the paper
“Droplet doublets could also appear as multiple barcode calls, but the predicted doublet rate of ~2% (Zheng et al., 2017) would account for only a fraction of the cells with more than one barcode.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
What the sources say
- Not checked yetcites Zheng et al., 2017 source
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.
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
“A UMAP representation of the single cell transcriptomes showed a high degree of overlap between cells from two human donors and marked differences between the ex vivo (input) cell population and tumor infiltrating (in vivo) T cells including differential expression of genes associated with proliferation or effector function (Figure 6C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
plaincorrelationaln = 2- 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
“Over 40,000 individual cells were sequenced, of which ~58% were successfully assigned one (monoallelic) or two (biallelic) barcodes (Figure 5F).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plainn = 2- 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
“T cells engineered with a polycistronic knock-in construct expressing a NY-ESO-1 specific TCR (1G4 clone) along with either a control construct (tNGFR), the transcription factor TCF7, or the chimeric TGFβR2–41BB receptor all showed statistically significant reductions in tumor size relative to vehicle only injections of media (Figure 7B).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > A novel TGFβR2–41BB chimeric receptor nominated by in vitro and in vivo pooled knock-in screens improves solid tumor clearance by primary human T cells
plaindirectn = 4 ·- 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
“While both the transcription factor TCF7 and TGFβR2-derived synthetic receptors increased T cell abundance in solid tumors, they induced different cell states.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
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
“Our integrated pooled knock-in platform nominated a lead hit (TGFβR2–41BB) that increased T cell fitness in vivo and promoted key effector cytokines including IFNɣ.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
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
“Surface and intracellular staining of cells with eight individual knock-in constructs revealed robust expression for each expected protein, including increased expression of Fas and CD25 (IL2RA) above endogenous expression levels in stimulated T cells (Figure 4B and Figure S5A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in hits individually validated and improved in vitro cancer cell killing
plaindirectn = 8- 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
“Nonetheless, across four individual human donors, several knock-in constructs promoted statistically significant in vivo accumulation of engineered T cells in the tumor environment (Figure 6B), including a construct not identified in the in vitro screens.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
plaindirectn = 4- 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
“Next-generation T cell therapies are being engineered with transgenes designed to overcome suppressive signals in the tumor environment (Kloss et al., 2018; Lynn et al., 2019; Yamamoto et al., 2019).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
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
“Antigen-specific human T cells can be generated ex vivo by non-targeted viral transduction of defined T cell receptors (TCRs), chimeric antigen receptors (CARs), or other synthetic receptors (Esensten et al., 2017).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
- Not checked yetcites Esensten et al., 2017 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.
In the paper
“Fas is a TNF super family surface receptor that mediates apoptosis through interactions with its ligand, FASL (Strasser et al., 2009).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in screens identify gene constructs to improve context-dependent fitness of human T cells
What the sources say
- Not checked yetcites Strasser et al., 2009 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.
None of the sources cited for this claim have been checked yet.
In the paper
“CRISPR-Cas9 genetic ablation of checkpoint targets such as PD-1 in adoptive T cell therapies has also shown increased functionality in certain models and is being persued clinically (Ren et al., 2017; Rupp et al., 2017; Stadtmauer et al., 2020; Su et al., 2016).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
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. 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
“These results showed large pooled knock-in libraries can be generated and their barcodes can be quantitatively sequenced in a human T cell population.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > Pooled knock-in of a multiplexed library of large DNA constructs
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.
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“Genetically engineered immune cell therapies have proven effective for treatment of some cancer types (Esensten et al., 2017; Fesnak et al., 2016; June and Sadelain, 2018; Sadelain et al., 2017), but they still cannot cure most tumors.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > INTRODUCTION
What the sources say
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.
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
“These data suggest that TCF7 promotes a more naïve or quiescent state, whereas the TGFβR2–41BB construct promotes accumulation of cells expressing key effector cytokine genes.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
hedgeddirectn = 2- 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
“This allowed us not only to screen for constructs that increased cell abundance with different in vitro and in vivo selective pressures, but also to perform high dimensional analysis of the cellular phenotypes resulting from each large knock-in DNA cassette.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
plaindirect- Asserted: passes — The authors put it forward as true.
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- 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
“Consistent with this model, in mouse chronic viral infection and syngeneic tumor models, TCF7+ cells have increased proliferative potential and less differentiated phenotypes during immunotherapies (Kurtulus et al., 2019; Siddiqui et al., 2019; Wu et al., 2016)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
What the sources say
plaincorrelational- 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
“In contrast to these technologies that have shown promise for screening small mutations in immortalized cells lines, we now present a new CRISPR platform to test large knock-in candidate therapeutic constructs in primary human cells, which should accelerate the development of improved cellular therapies.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
hedgedconditional- 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: Technical challenges have limited CRISPRa applications in therapeutically relevant primary human cell types.
- Rests on an assumption: CRISPRa can only induce natural gene products encoded in the genome and does so with variable efficiency.
None of the sources cited for this claim have been checked yet.
In the paper
“These vectors have variable integration copy numbers and integrate at semi-random genome sites, which can cause variability in transgene expression and altered cell phenotypes due to insertional mutagenesis (Cavazzana-Calvo et al., 2010; Romero et al., 2013).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
What the sources say
- Not checked yetcites Cavazzana-Calvo et al., 2010 source
- Not checked yetcites Romero et al., 2013
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.
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
“Quality control metrics following PoKI-Seq confirmed robust rates of gene and UMI identification per cell and an average >130X coverage of cells assigned to each knock-in construct (per blood donor and per ex vivo condition tested) (Figure S6B–C).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
plainn = 2- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
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- 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
“TGFβ is a key suppressive cytokine normally produced by immune cells but which can be co-opted by tumors to suppress effective immune responses (Batlle and Massagué, 2019; Gigante et al., 2012).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
What the sources say
- Not checked yetcites Batlle and Massagué, 2019
- Not checked yetcites Gigante et al., 2012 source
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 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
“Barcoded knock-in libraries have even broader potential to study functional effects of sequence variation at any selected site in the genome.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
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 contrast to lentiviral and retroviral transgenesis, pooled non-viral HDR templates allow for targeted integration at a defined site with relatively consistent transgene expression levels and low rates of barcode mis-assignment without requiring viral packaging steps.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
It may state two separate claims as one.
- Rests on an assumption: A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.
In the paper
“In addition, lentiviral and retroviral library preparation is laborious and can lead to significant barcode swapping due to viral recombination (Sack et al., 2016).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > DISCUSSION
What the sources say
- Not checked yetcites Sack et al., 2016 source
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.
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).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
What the sources say
- Not checked yetcites Butler et al., 2018 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. 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
“Fresh blood was taken from healthy human donors under a protocol approved by the UCSF Committee on Human Research (CHR #13–11950).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > EXPERIMENTAL MODEL AND SUBJECT DETAILS
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.
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
“With the parameters of the pooled knock-in library design (~400 bps between unique library insert and its corresponding barcode, separated by the new knocked in TCR-α specificity) described here (Figure 1B), the amount of predicted template switching with pooled assembly reactions was ~50%, and with a pooled electroporation condition was only ~10%.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
It may state two separate claims as one.
- The authors state this as an assumption: Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.
- The authors state this as an assumption: A template that has switched once is equally likely to switch again.
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
“Only cells with more than 500 genes identified were used for downstream analyses.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
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 animal experiments were performed in compliance with relevant ethical regulations per an approved IACUC protocol (UCSF), including a tumor size limit of 2.0 cm in any dimension.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > EXPERIMENTAL MODEL AND SUBJECT DETAILS
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.
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
“Given 1000 sorted knock-in positive cells, 75% will possess a monoallelic knock-in (750 cells, 750 knock-in alleles), while 25% will possess biallelic knock-ins (250 cells, 500 knock-in alleles).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
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
“Investigators were blinded to experimental treatment group during sizing measurements.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > EXPERIMENTAL MODEL AND SUBJECT DETAILS
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
“We filtered out reads appearing less than twice and cells with more than two assigned knock-in barcodes.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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. 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
“In a two-member library half of the actual template switching will occur with another template that contains the same barcode, and is thus not detected. Therefore, the observed amount of switching must be multiplied by 2.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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.
It may state two separate claims as one.
- The authors state this as an assumption: Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.
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 statistical analysis to account for biological noise.”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > 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: 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 relies on this without support.
The field lacks rapid methods to test and directly compare the functions of large DNA knock-in integrations in human primary cells.
“However, to date we have lacked rapid methods to test and directly compare the functions of large DNA knock-in integrations in human primary cells, which would accelerate the development of more effective cell therapies.”
The paper relies on this without support.
Cell abundance alone is insufficient to assess cellular function, and an ideal screening method should also assess modified cell states.
“However, cell abundance measures only one aspect of cellular function, and an ideal screening methodology would allow systematic assessment of modified cell states as well.”
The paper relies on this without support.
The in vitro conditions used in the pooled screens mimic aspects of tumor microenvironments.
“we repeated the pooled screen in diverse in vitro conditions designed to mimic aspects of tumor micro-environments”
The paper relies on this without support.
CAR T cells have not proved effective for solid tumors.
“CAR T cells are now approved therapy for some hematological malignancies, but have not proved effective for solid tumors.”
The paper relies on this without support.
For many clinical applications, engineering antigen specificity alone will not be sufficient to cure disease.
“Mounting evidence suggests that for many clinical applications engineering antigen specificity will not be sufficient to cure disease.”
The paper relies on this without support.
T cell function is often suppressed by tumors, and further engineering of antigen-specific T cells is required to promote more effective cell states.
“For example, T cell function is often suppressed by tumors and further engineering of antigen-specific T cells will be required to promote more effective cell states.”
The paper relies on this without support.
The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.
“The field needs a systematic approach to compare complex transgene constructs head-to-head and determine which are most effective for specific disease indications.”
The paper relies on this without support.
Technical challenges have limited CRISPRa applications in therapeutically relevant primary human cell types.
“Technical challenges have limited CRISPRa applications in therapeutically relevant primary human cell types.”
The paper relies on this without support.
CRISPRa can only induce natural gene products encoded in the genome and does so with variable efficiency.
“Furthermore, CRISPRa can only induce natural gene products encoded in the genome and does so with variable efficiency.”
The paper relies on this without support.
A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.
“A major challenge to quantitative tracking of targeted genomic integrations of pooled knock-in constructs has been the abundance of non-integrated DNA templates (viral or non-viral) in targeted cells and the risk of template switching during molecular steps of pooled library production, integration, and sequencing.”
The paper relies on this without support.
Most over-expression studies have been performed with lentiviral or retroviral vectors.
“Most over-expression studies have been performed with lentiviral or retroviral vectors.”
The authors state this as an assumption.
Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.
“Assumption 1: Template switching is random, and any barcode that is swapped is equally likely to switch to any other barcode.”
The authors state this as an assumption.
A template that has switched once is equally likely to switch again.
“Assumption 2: A template that has switched once is equally likely to switch again.”
The authors state this as an assumption.
The knock-in library is arbitrarily large.
“assuming an arbitrarily large knock-in library”
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
“PoKI-seq [14], combines single-cell transcriptome analysis and pooled knockin screening.”
From A mini-review on perturbation modelling across single-cell omic modalities (2024) · cites this paper as [14]
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 major: it would touch a result — a value a result rests on, reported and recomputed p-values on opposite sides of the significance level, two passages that conflict with no sign which is right, or a cited work retracted before this paper was published.
In the paper
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > METHOD DETAILS
“The control T cell population clearance value was divided by the experimental T cell population clearance value”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > METHOD DETAILS
“an A375 clearance value greater than 1 indicates more cancer cell killing relative to control T cells, while a value less than 1 indicates decreased cancer cell killing relative to control T cells.”
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
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > METHOD DETAILS
“Single-cell RNA sequencing was performed on 8 separate samples (2 donors, 2 recipients per donor, matched pre- and post-implantation cells) with the Chromium Single Cell 3′ Reagent Kit, v3 chemistry (10x Genomics, PN-1000092)”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > STAR Methods > QUANTIFICATION AND STATISTICAL ANALYSIS
“Pre-processing of the Illumina sequencing results from the 10X Genomics V2 libraries was performed with CellRanger software, version 2.0.0.”
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
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > In vivo PoKI-seq of engineered human T cells in a solid tumor microenvironment
“PoKI-Seq with combined single cell amplicon and RNA-sequencing (Figure 4A).”
§ Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies > RESULTS > PoKI-Seq: Pooled knock-ins with single-cell transcriptome analysis to assess abundance and cell state
“We next tested whether pooled knock-in screening could similarly be coupled with scRNA-seq to generate high-dimensional phenotypic information on modified cell states while also recording each cell’s specific knock-in construct barcodes, a method we term PoKI-Seq (Pooled Knock-In Sequencing) (Figure 5A).”
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
Massively parallel digital transcriptional profiling of single cells
Zheng, G. X. Y., Terry, J. M., Belgrader, P., Ryvkin, P., Bent, Z. W., Wilson, R., Ziraldo, S. B., Wheeler, T. D., McDermott, G. P., Zhu, J., et al. (2017) · Nature Communications · doi:10.1038/ncomms14049 · Cited by 7,353
Oncology meets immunology: the cancer-immunity cycle.
Chen, D. S., & Mellman, I. (2013) · Immunity · doi:10.1016/j.immuni.2013.07.012 · Cited by 6,929
Understanding the tumor immune microenvironment (TIME) for effective therapy
Binnewies, M., Roberts, E. W., Kersten, K., Chan, V., Fearon, D. F., Merad, M., Coussens, L. M., Gabrilovich, D. I., Ostrand-Rosenberg, S., Hedrick, C. C., et al. (2018) · Nature Medicine · doi:10.1038/s41591-018-0014-x · Cited by 6,145
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
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
Wherry, E. J. (2011) · Nature Immunology · doi:10.1038/ni.2035 · Cited by 4,062
Fast gene set enrichment analysisPreprint
Korotkevich, G., Sukhov, V., Budin, N., Shpak, B., Artyomov, M. N., & Sergushichev, A. (2016) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/060012 · Cited by 3,742
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., et al. (2013) · Cell · doi:10.1016/j.cell.2013.06.044 · Cited by 3,696
Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma
Zaretsky, J. M., Garcia-Diaz, A., Shin, D. S., Escuin-Ordinas, H., Hugo, W., Hu-Lieskovan, S., Torrejon, D. Y., Abril-Rodriguez, G., Sandoval, S., Barthly, L., et al. (2016) · New England Journal of Medicine · doi:10.1056/nejmoa1604958 · Cited by 3,237
Cited by
Sorted by citations
CD8+ T cell differentiation and dysfunction in cancer
Philip, M., & Schietinger, A. (2022) · Nature Reviews Immunology · doi:10.1038/s41577-021-00574-3 · Cited by 1,052
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
Targeting the epigenetic regulation of antitumour immunity
Hogg, S. J., Beavis, P. A., Dawson, M. A., & Johnstone, R. W. (2020) · Nature Reviews Drug Discovery · doi:10.1038/s41573-020-0077-5 · Cited by 665
Engineering CAR-T Cells for Next-Generation Cancer Therapy
Hong, M., Clubb, J. D., & Chen, Y. Y. (2020) · Cancer Cell · doi:10.1016/j.ccell.2020.07.005 · Cited by 659
Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1-CD8+ Tumor-Infiltrating T Cells
Kurtulus, S., Madi, A., Escobar, G., Klapholz, M., Nyman, J., Christian, E., Pawlak, M., Dionne, D., Xia, J., Rozenblatt-Rosen, O., et al. (2019) · Immunity · doi:10.1016/j.immuni.2018.11.014 · Cited by 636
Metadata
- Authors
- Roth, T. L., Li, P. J., Blaeschke, F., Nies, J. F., Apathy, R., Mowery, C., Yu, R., Nguyen, M. L. T., Lee, Y., Truong, A., Hiatt, J., Wu, D., Nguyen, D. N., Goodman, D., Bluestone, J. A., Ye, C. J., Roybal, K., Shifrut, E., & Marson, A.
- Affiliations
- University of California, San Francisco (US)
- Corresponding author
- Roth, Theodore L.
- Marson, Alexander
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2020-04
- Volume
- 181
- Issue
- 3
- Pages
- 728-744.e21
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC7219528
- OpenAlex
- W3017040524
- Semantic Scholar
- fef922b8a02fcf158d7a3465ed812a49ef0538b1
- Cited by
- 186
- References
- 77
- Citations by year
- 2026: 11 · 2025: 23 · 2024: 25 · 2023: 29 · 2022: 45 · 2021: 32 · 2020: 19
- FWCI
- 10.13
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health
- 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 › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- T cell engineering, adoptive immunotherapy, barcoding, single-cell transcriptomics, cancer immunotherapy
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Animals, Blood Cells, Genetic Engineering, Humans, Immunotherapy, T-Lymphocytes, Mice, SCID, Mice, Inbred NOD, Mice, Gene Knock-In Techniques, Single-Cell Analysis, Transcriptome, 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.
A language model drew the claims from the paper on 2026-10-05, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-10-05 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Cited sources are checked with a language model’s help (gpt-6-sol).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
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