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Genome-scale CRISPR screening for modifiers of cellular LDL uptake
Open accessPublished version of a preprint
Abstract
Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.
Overview
AI snapshot
Written from the paper’s full text.
TL;DR
Genome-scale and focused CRISPR screens in HuH7 cells identified 118 positive and 45 negative regulators of cellular LDL uptake, many not previously linked to cholesterol regulation. Orthogonal screens showed most hits were not general endocytosis defects, a subset acted independently of LDLR, and exocyst disruption reduced LDL but increased transferrin uptake.
Key findings
- Primary genome-wide screen (GeCKOv2, 123,411 gRNAs) found 95 candidate genes with FDR<5% whose targeting reduced LDL uptake under lipoprotein-rich or lipoprotein-depleted conditions; only SQLE was associated with enhanced LDL uptake.
- Secondary custom screen (12,207 gRNAs, 15 per gene) identified 118 positive and 45 negative regulators of HuH7 LDL uptake at FDR<5%, with 66 positive and 18 negative regulators significant under both lipoprotein-rich and lipoprotein-depleted conditions.
- Among 163 validated LDL uptake regulators, 12% (20/163) had a genome-wide significant LDL GWAS result within the gene and 33% (54/163) within 500 kb; p-values within these genes were more significant than length-matched controls (two-sided p < 2.2×10⁻¹⁶).
- In LDLR-deleted HuH7 cells, 45/118 positive and 17/45 negative regulators showed similar effects; EXOC4 or EXOC8 disruption caused ~40% decreased LDL uptake and ~2–3 fold increased transferrin uptake, rescued by CRISPR-resistant cDNA.
Methods
- Pooled CRISPR screens in HuH7 cells: primary genome-wide GeCKOv2 screen at ~25 million cells and 200× gRNA coverage, 13-day expansion, 1 day lipoprotein-rich or lipoprotein-depleted culture, fluorescent LDL uptake, FACS sorting top/bottom 7.5%, MAGeCK analysis; three biologic replicates per screen.
- Secondary custom gRNA library targeting 554 positive and 170 negative candidate genes with 15 gRNAs per gene plus 1000 non-targeting controls; orthogonal screens tested transferrin uptake, LDLR-deleted HuH7 cells, surface/total LDLR staining, and HepG2 LDL uptake.
- GWAS comparison used SAIGE in 388,629 white British UK Biobank individuals for LDL cholesterol; functional annotation used PANTHER Gene Ontology overrepresentation.
Limitations
- Stated: screens used immortalized hepatoma cells in two-dimensional culture, so in vivo relevance is uncertain; the fluorescent LDL tracer was NHS-ester-labeled and may not fully reflect native LDL dependencies; FDR thresholds are somewhat arbitrary, likely yielding some false positives and false negatives.
- Stated: genes that are essential or confer a fitness advantage may be missed due to gRNA depletion; redundant genes may be missed, e.g., SREBF1/SREBF2; only cell-autonomous effects are detectable, e.g., PCSK9 secreted by neighboring cells can mask mutant phenotypes.
- Evident from the text: strong cell-type specificity was observed, with limited overlap between HuH7 and HepG2 hits and only 1 gene shared with a prior endothelial cell siRNA screen, so generalizability across contexts requires empirical testing.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
16 of 35 cited sources checked · 9 supported · 1 flagged · accuracy not yet measured
- Findingown data
Comparison of the authors' data to a previous siRNA screen for endothelial cell LDL uptake revealed limited overlap, with only 1 gene identified in both studies.
“Comparison of our data to a previous siRNA screen for endothelial cell LDL uptake[19] revealed limited overlap, with only 1 gene identified in both studies (S9 Table).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [19] JR Kraehling, 2016, Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells, Nat Commun source
- Findingown data
Most targeted genes exhibiting decreased LDLR staining (surface or total cell-associated) had also exhibited decreased fluorescent LDL uptake.
“Most targeted genes exhibiting decreased LDLR staining (surface or total cell-associated) had also exhibited decreased fluorescent LDL uptake (Fig 5E).”
Quote in the paperSubstantial claim - Findingown data
The likelihood of a gene showing a functional influence on LDL uptake by HepG2 cells was predicted by the strength of its association with LDL uptake by HuH7 cells.
“The likelihood of a gene showing a functional influence on LDL uptake by HepG2 cells was predicted by the strength of its association with LDL uptake by HuH7 cells (Fig 6G).”
Quote in the paperSubstantial claim - Findingown data
TFRC was the top hit whose disruption was associated with reduced transferrin uptake.
“As expected, TFRC was the top hit whose disruption was associated with reduced transferrin uptake (Fig 3A and S6 Table).”
Quote in the paperNot a substantial claim - Findingown data
Under lipoprotein-depleted conditions, disruption of 53 and 5 genes was associated with reduced or increased LDL uptake in HepG2 cells, respectively.
“A much higher number of LDL uptake modifiers were identified under lipoprotein-depleted conditions, with disruption of 53 and 5 genes associated with reduced or increased LDL uptake, respectively (Fig 6D volcano).”
Quote in the paperSubstantial claim
- Findingown data
LDL uptake by HuH7 cells was readily detectable above cellular autofluorescence by flow cytometry following a 1 hour incubation with 4 μg/mL of fluorescently-conjugated LDL in serum-free media.
“LDL uptake by HuH7 cells was readily detectable above cellular autofluorescence by flow cytometry following a 1 hour incubation with 4 μg/mL of fluorescently-conjugated LDL in serum-free media (S1A Fig).”
Quote in the paperSubstantial claim - Findingown data
A high degree of concordance was observed for identified positive regulators between the screens conducted under lipoprotein-rich or lipoprotein-depleted conditions, with 27/95 hits identified under both conditions.
“A high degree of concordance was observed for identified positive regulators between the screens conducted under lipoprotein-rich or lipoprotein-depleted conditions, with 27/95 hits identified under both conditions (S2 Fig).”
Quote in the paperNot a substantial claim - Findingown data
Screening for total LDLR revealed 46 and 43 genes whose targeting either reduced or enhanced total cellular LDLR staining (FDR<0.05).
“Screening for total LDLR similarly revealed 46 and 43 genes whose targeting either reduced or enhanced total cellular LDLR staining (FDR<0.05, Fig 5C).”
Quote in the paperSubstantial claim - Findingown data
Screen hits exhibited a clearer discrimination from genes with no signal in the secondary screen.
“Screen hits exhibited a clearer discrimination from genes with no signal (Fig 2H).”
Quote in the paperSubstantial claim - Findingown data
Ontology analysis of validated HuH7 LDL uptake regulators revealed significant enrichment for several annotations including genes involved in regulation of gene expression, cholesterol metabolism, Golgi to plasma membrane transport, protein N-linked glycosylation, and ubiquitin-mediated protein degradation.
“Ontology analysis of our validated HuH7 LDL uptake regulators revealed significant enrichment for several annotations including genes involved in regulation of gene expression, cholesterol metabolism, Golgi to plasma membrane transport, protein N-linked glycosylation, and ubiquitin-mediated protein degradation (S6 Fig and S4 Table).”
Quote in the paperNot a substantial claim - Findingown data
A total of 163 genes were identified to be either positive or negative regulators of HuH7 LDL uptake.
“A total of 163 genes were identified to be either positive or negative regulators of HuH7 LDL uptake.”
Quote in the paperSubstantial claim - Findingown data
Gene targeted cells with increased surface or total LDLR exhibited heterogeneous effects on LDL uptake, with roughly equal numbers exhibiting either reduced or increased LDL uptake.
“In contrast, gene targeted cells with increased surface or total LDLR exhibited heterogeneous effects on LDL uptake, with roughly equal numbers exhibiting either reduced or increased LDL uptake (Fig 5E).”
Quote in the paperSubstantial claim - Findingown data
Among the 95 candidate genes were known regulators of LDL uptake including LDLR, SCAP, and MBTPS1.
“Among these candidates were known regulators of LDL uptake including LDLR, SCAP, and MBTPS1, though MBTPS2 was not identified among the screen hits (ranking 343 and 58 under lipoprotein-rich and lipoprotein-depleted conditions, with FDR>5% for both).”
Quote in the paperNot a substantial claim - Findingown data
Only 1 gene, SQLE, was identified whose targeting was associated with enhanced LDL uptake.
“Only 1 gene, SQLE, was identified whose targeting was associated with enhanced LDL uptake (Fig 1D and 1E and S1, S2 Tables).”
Quote in the paperSubstantial claim - Findingown data
Through a primary genome-wide CRISPR screen followed by a focused secondary gRNA library with greater depth of coverage, the authors validated 118 positive regulators and 45 negative regulators of HuH7 cell LDL uptake.
“Through a primary genome-wide CRISPR screen, followed by the synthesis and re-screening of a focused secondary gRNA library with greater depth of coverage, we validated 118 positive regulators and 45 negative regulators of HuH7 cell LDL uptake.”
Quote in the paperSubstantial claim - Findingown data
Genes positively identified under lipoprotein-depleted conditions were 384-fold more likely than negative genes to also be identified under lipoprotein-rich conditions.
“Genes positively identified under lipoprotein-rich conditions were 246-fold more likely than negative genes to also be identified under lipoprotein-depleted conditions; genes positively identified under lipoprotein-depleted conditions were 384-fold more likely than negative genes to also be identified under lipoprotein-rich conditions.”
Quote in the paperSubstantial claim - Findingown data
P-values for association with LDL cholesterol within the 163 identified genes were more significant, on average, than those within length-matched control genes.
“P-values for association with LDL cholesterol within the 163 identified genes were also more significant, on average, than those within length-matched control genes (two-sided p-value < 2.2x10-16, Fig 2J and S5 Table).”
Quote in the paperSubstantial claim - Findingown data
The majority of LDLR-independent LDL uptake regulators (44/79 positive regulators, 18/31 negative regulators) were identified under both lipoprotein-rich and lipoprotein-depleted conditions.
“The majority of LDLR-independent LDL uptake regulators (44/79 positive regulators, 18/31 negative regulators) were identified under both lipoprotein-rich and lipoprotein-depleted conditions (S7A and S7B Fig).”
Quote in the paperSubstantial claim - Findingown data
Acquisition of fluorescent signal was mediated by LDLR, as CRISPR-mediated targeting of LDLR resulted in a ~75% reduction in LDL uptake under the assay conditions.
“Acquisition of fluorescent signal was mediated by LDLR, as CRISPR-mediated targeting of LDLR resulted in a ~75% reduction in LDL uptake under these conditions (S1D Fig).”
Quote in the paperSubstantial claim - Findingown data
Genes positively identified under lipoprotein-rich conditions were 246-fold more likely than negative genes to also be identified under lipoprotein-depleted conditions.
“Genes positively identified under lipoprotein-rich conditions were 246-fold more likely than negative genes to also be identified under lipoprotein-depleted conditions; genes positively identified under lipoprotein-depleted conditions were 384-fold more likely than negative genes to also be identified under lipoprotein-rich conditions.”
Quote in the paperSubstantial claim - Findingown data
Little concordance was observed between regulators of LDL and transferrin uptake.
“Little concordance was observed between regulators of LDL and transferrin uptake (Fig 3B–3D).”
Quote in the paperSubstantial claim - Findingown data
The LDLR-deleted HuH7 clone exhibited a ~85% reduction in LDL uptake relative to parental wild-type cells.
“To test this hypothesis, we generated an HuH7 clone harboring a homozygous frameshift mutation in LDLR (Fig 4A), with no detectable LDLR protein by immunoblotting (Fig 4B) and a ~85% reduction in LDL uptake relative to parental wild-type cells (Fig 4C).”
Quote in the paperSubstantial claim - Findingown data
Among the 736 genes tested with the secondary library, 24 were found to positively regulate and 19 to negatively regulate transferrin uptake (FDR<0.05).
“Among the 736 genes tested with our secondary library, 24 were found to positively regulate and 19 to negatively regulate transferrin uptake (FDR<0.05).”
Quote in the paperSubstantial claim - Findingown data
Comparison to current human GWAS data from UK Biobank showed a significant enrichment for genes in proximity to genetic variants associated with LDL cholesterol relative to matched control genes.
“Comparison to current human GWAS data from UK Biobank showed a significant enrichment for genes in proximity to genetic variants associated with LDL cholesterol relative to matched control genes.”
Quote in the paperSubstantial claim - Findingown data
Gene-level analysis identified 95 candidate genes with a false discovery rate (FDR) <5% whose targeting was associated with reduced LDL uptake under either lipoprotein-rich or lipoprotein-deficient conditions.
“Gene-level analysis identified 95 candidate genes with a false discovery rate (FDR) <5% whose targeting was associated with reduced LDL uptake under either lipoprotein-rich or lipoprotein-deficient conditions (Figs 1B, 1C and S1, S2 Tables).”
Quote in the paperSubstantial claim - Findingown data
The validation rate of candidates in the secondary screen was strongly correlated to the strength of signal in the primary screen.
“The validation rate of candidates in the secondary screen was strongly correlated to the strength of signal in the primary screen (Fig 2E).”
Quote in the paperSubstantial claim - Findingown data
Ontology analysis of genes identified under either condition revealed enrichment in multiple annotations including cholesterol biosynthesis, metabolism, and vesicular trafficking.
“Ontology analysis of genes identified under either condition revealed enrichment in multiple annotations including cholesterol biosynthesis, metabolism, and vesicular trafficking (S7C Fig).”
Quote in the paperSubstantial claim - Findingown data
The authors identified 26 and 20 genes whose targeting either reduced or enhanced LDLR surface staining, respectively (FDR<0.05).
“We identified 26 and 20 genes whose targeting either reduced or enhanced LDLR surface staining, respectively (FDR<0.05, Fig 5A).”
Quote in the paperSubstantial claim - Findingown data
The concordance between screen conditions extended to the individual gRNA level, as the relative ranking and magnitude of enrichment for individual gRNAs under lipoprotein-rich conditions was correlated with their corresponding value under lipoprotein-depleted conditions.
“This concordance between screen conditions also extended to the individual gRNA level, as the relative ranking (Fig 2G) and magnitude of enrichment (S5C Fig) for individual gRNAs under lipoprotein-rich conditions was correlated with their corresponding value under lipoprotein-depleted conditions.”
Quote in the paperNot a substantial claim - Findingown data
Among the candidate LDL uptake modifiers identified in the primary CRISPR screen were multiple components of the exocyst.
“Among the candidate LDL uptake modifiers identified in our primary CRISPR screen were multiple components of the exocyst, an octameric protein complex involved in vesicular trafficking[20].”
Quote in the paperSubstantial claim - Findingreasoning
We did not detect significant effects upon disruption of SREBF1 or SREBF2.
“we did not detect significant effects upon disruption of SREBF1 or SREBF2”
Quote in the paperSubstantial claim - Findingown data
Screening of LDL uptake in a LDLR-deleted clone confirmed that many of our screen hits were dependent on LDLR for their functional influence on LDL uptake.
“Screening of LDL uptake in a LDLR-deleted clone confirmed that many of our screen hits were dependent on LDLR for their functional influence on LDL uptake.”
Quote in the paperSubstantial claim - Findingown data
Disruption of SCARB1 was associated with reduced LDL uptake in both WT and LDLR-deleted HuH7 cells.
“Supporting this model, we found disruption of SCARB1 to be associated with reduced LDL uptake in both WT and LDLR-deleted HuH7 cells.”
Quote in the paperSubstantial claim - Findingown data
We detected Vps29 and CCDC22 as positive regulators of LDL uptake in HuH7 cells.
“we did detect some of these components (Vps29, CCDC22) as positive regulators of LDL uptake in HuH7 cells.”
Quote in the paperSubstantial claim - Findingown data
Our validation rate of hits was highly dependent on the strength of signal for a candidate gene in the primary screen, demonstrating a significant correlation over independent experiments.
“Second, our validation rate of hits was highly dependent on the strength of signal for a candidate gene in the primary screen, demonstrating a significant correlation over independent experiments.”
Quote in the paperSubstantial claim - Findingown data
Additional genes consistently identified across our screens included the positive control genes SCAP, MBTPS1, and MBTPS2.
“Additional genes consistently identified across our screens included the positive control genes SCAP, MBTPS1, and MBTPS2.”
Quote in the paperSubstantial claim - Findingown data
Disruption of each of the 6 exocyst genes targeted in the secondary CRISPR library was associated with a significant reduction (FDR<5%) of LDL uptake in HuH7 cells tested under either lipoprotein-rich or lipoprotein-depleted culture conditions.
“Disruption of each of the 6 exocyst genes targeted in the secondary CRISPR library was associated with a significant reduction (FDR<5%) of LDL uptake in HuH7 cells tested under either lipoprotein-rich or lipoprotein-depleted culture conditions (S2 Table).”
Quote in the paperSubstantial claim - Findingnone given
Our screen hits exhibit a high degree of cell type specificity.
“The high degree of cell type specificity for our screen hits emphasizes the need for empirical testing of identified genes in other contexts.”
Quote in the paperNot a substantial claim - Findingown data
All 6 tested subunits of the exocyst caused opposing effects on transferrin uptake.
“This latter group includes all 6 tested subunits of the exocyst”
Quote in the paperNot a substantial claim - Findingnone given
Exocyst disruption in WT cells results in a ~40% reduction in LDL uptake.
“the ~40% reduction in LDL uptake resulting from exocyst disruption in WT cells”
Quote in the paperSubstantial claim - Findingown data
Each of the 6 exocyst genes was associated with an opposite effect on the uptake of transferrin.
“Intriguingly, each of these 6 exocyst genes was associated with an opposite effect on the uptake of transferrin (Fig 7A), suggestive of cargo selectivity rather than a global influence on endocytosis or receptor recycling.”
Quote in the paperSubstantial claim - Findingown data
The concordance between conditions extended to the individual gRNA level, as gRNAs showing significant activity for one condition were much more likely to show activity for the other condition.
“This concordance also extended to the individual gRNA level, as gRNAs showing significant activity for one condition were much more likely to show activity for the other condition.”
Quote in the paperSubstantial claim - Findingown data
SCAP, MBTPS1, and MBTPS2 influenced LDL uptake on a LDLR-deleted genetic background.
“These included SCAP, MBTPS1, and MBTPS2”
Quote in the paperSubstantial claim - Findingown data
Our screen hits exhibited a high degree of concordance between lipoprotein-rich and lipoprotein-depleted conditions, much greater than might be expected by stochastic variation alone.
“Third, our screen hits exhibited a high degree of concordance between lipoprotein-rich and lipoprotein-depleted conditions, much greater than might be expected by stochastic variation alone.”
Quote in the paperSubstantial claim - Findingown data
Disruption of the exocyst was also associated with reduced LDL uptake on a LDLR-deleted background.
“Similarly, disruption of the exocyst was also associated with reduced LDL uptake on a LDLR-deleted background.”
Quote in the paperSubstantial claim - Findingown data
We observed a high degree of concordance between orthogonal screens.
“we also observed a high degree of concordance between orthogonal screens.”
Quote in the paperSubstantial claim - Findingnone given
LDL uptake in LDLR-deleted cells is only ~15% of that in WT cells.
“LDL uptake in LDLR-deleted cells is only ~15% of that in WT cells.”
Quote in the paperNot a substantial claim - Findingown data
Functional annotations of our novel screen hits showed modest enrichment in some pathways, including N-glycosylation, ubiquitination, and transcriptional regulation.
“Functional annotations of our novel screen hits showed modest enrichment in some pathways, including N-glycosylation, ubiquitination, and transcriptional regulation.”
Quote in the paperNot a substantial claim - Findingown data
Genome-wide CRISPR screening identified a large set of genes that modify cellular LDL uptake, many not previously recognized to play a role in LDL uptake.
“We applied genome-wide CRISPR screening to identify novel determinants of cellular LDL uptake, identifying a large set of genes, many of which were not previously recognized to play a role in LDL uptake.”
Quote in the paperSubstantial claim - Findingown data
Many LDL uptake regulators did not exhibit a readily detectable impact on LDLR levels either at the cell surface or associated with the entire cell.
“We found that many LDL uptake regulators did not exhibit a readily detectable impact on LDLR levels either at the cell surface or associated with the entire cell”
Quote in the paperNot a substantial claim - Findingnone given
The regions containing the identified genes were enriched for significant associations with LDL in a genome-wide association study of nearly 400,000 Europeans.
“In addition, the regions containing the identified genes were enriched for significant associations with LDL in a genome-wide association study of nearly 400,000 Europeans.”
Quote in the paperSubstantial claim - Findingown data
Significant positive correlation was observed for the degree of enrichment for a given LDL uptake modifier in either HuH7 or HepG2 cells.
“Significant positive correlation was observed for the degree of enrichment for a given LDL uptake modifier in either cell line (Fig 6H).”
Quote in the paperSubstantial claim - Findingown data
Several LDL uptake modifiers also seemed to influence LDL uptake on a LDLR-deleted genetic background.
“Several LDL uptake modifiers however also seemed to influence LDL uptake on a LDLR-deleted genetic background.”
Quote in the paperSubstantial claim - Findingown data
Genes whose perturbation impacted LDL uptake were much more likely to also be associated with reduced surface or total LDLR abundance.
“For example, genes whose perturbation impacted LDL uptake were much more likely to also be associated with reduced surface or total LDLR abundance.”
Quote in the paperSubstantial claim - Findingown data
No genes were identified that associated with significant effects on LDL uptake in opposite directions in HuH7 and HepG2 cells.
“No genes were identified that associated with significant effects on LDL uptake in opposite directions in HuH7 and HepG2 cells.”
Quote in the paperSubstantial claim - Interpretationown data
A significant subset of the LDL uptake modifiers identified may influence LDLR-independent, or both LDLR-dependent and independent, LDL uptake.
“Instead, these findings suggest that a significant subset of the LDL uptake modifiers identified here may influence LDLR-independent, or both LDLR-dependent and independent, LDL uptake.”
Quote in the paperSubstantial claim - Interpretationown data
The identification of LDLR-independent LDL uptake regulators is unlikely to be due to an influence on residual LDLR expression, as LDLR-targeting gRNAs were not enriched in LDLlow cells.
“The identification of these genes is unlikely to be due to an influence on residual LDLR expression, as LDLR-targeting gRNAs were not enriched in LDLlow cells.”
Quote in the paperSubstantial claim - Study design and conductown data
The LDLR-deleted HuH7 clone had no detectable LDLR protein by immunoblotting.
“To test this hypothesis, we generated an HuH7 clone harboring a homozygous frameshift mutation in LDLR (Fig 4A), with no detectable LDLR protein by immunoblotting (Fig 4B) and a ~85% reduction in LDL uptake relative to parental wild-type cells (Fig 4C).”
Quote in the paperSubstantial claim - Premisecitation
Only an estimated 20-30% of the heritability of lipid traits is currently explained.
“Moreover, only an estimated 20–30% of the heritability of lipid traits is currently explained[12].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [12] JS Dron, 2018, Polygenic influences on dyslipidemias, Curr Opin Lipidol source
- Interpretationreasoning
In accordance with its greater depth, the secondary screen exhibited more robust performance than the primary screen.
“In accordance with its greater depth (both in terms of gRNA per gene tested, and cells per gRNA tested), the secondary screen exhibited more robust performance than the primary screen.”
Quote in the paperSubstantial claim - Study design and conductown data
The authors generated an HuH7 clone harboring a homozygous frameshift mutation in LDLR.
“To test this hypothesis, we generated an HuH7 clone harboring a homozygous frameshift mutation in LDLR (Fig 4A), with no detectable LDLR protein by immunoblotting (Fig 4B) and a ~85% reduction in LDL uptake relative to parental wild-type cells (Fig 4C).”
Quote in the paperSubstantial claim - Interpretationnone given
The majority of the screen hits had not previously been implicated in cholesterol regulation.
“The majority of our screen hits had not previously been implicated in cholesterol regulation.”
Quote in the paperSubstantial claim - Interpretationown data
The majority of hits from the secondary screen do not appear to result from global disruption or stimulation of receptor-mediated endocytosis.
“Thus, the majority of hits from our secondary screen do not appear to result from global disruption or stimulation of receptor-mediated endocytosis.”
Quote in the paperSubstantial claim - Interpretationreasoning
It is likely that additional, as yet unrecognized genetic interactions contribute to cholesterol regulation in humans.
“It is therefore likely that additional, as yet unrecognized genetic interactions contribute to cholesterol regulation in humans.”
Quote in the paperSubstantial claim - Interpretationreasoning
A more limited gene list allows for greater depth of gRNA per gene, infected cells per gRNA, and sequencing reads per gRNA.
“A more limited gene list allows for greater depth of gRNA per gene, infected cells per gRNA, and sequencing reads per gRNA.”
Quote in the paperSubstantial claim - Interpretationown data
The opposite effects of exocyst disruption on LDL and transferrin uptake are suggestive of cargo selectivity rather than a global influence on endocytosis or receptor recycling.
“Intriguingly, each of these 6 exocyst genes was associated with an opposite effect on the uptake of transferrin (Fig 7A), suggestive of cargo selectivity rather than a global influence on endocytosis or receptor recycling.”
Quote in the paperSubstantial claim - Study design and conductreasoning
Our screen is limited in detecting only those genes which exhibit a phenotype through a cell-autonomous effect.
“Finally, our screen is limited in detecting only those genes which exhibit a phenotype through a cell-autonomous effect.”
Quote in the paperNot a substantial claim - Interpretationreasoning
The LDLR-dependence of uptake for our NHS-ester-labeled LDL tracer argues against a disruption of the ligand-receptor interaction.
“While the LDLR-dependence of uptake for this tracer argues against a disruption of the ligand-receptor interaction”
Quote in the paperNot a substantial claim - Interpretationcitation
The lack of detection of SREBF1 or SREBF2 effects is likely due to overlapping functions allowing one gene to compensate for loss of the other.
“likely due to overlapping functions allowing one gene to compensate for loss of the other[7]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [7] JD Horton, 2002, SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver, J Clin Invest source
- Interpretationreasoning
The presence of an LDLR-independent effect for a LDL uptake regulator does not rule out a concurrent LDLR-dependent mechanism.
“The presence of an LDLR-independent effect for a LDL uptake regulator does not rule out a concurrent LDLR-dependent mechanism.”
Quote in the paperNot a substantial claim - Interpretationreasoning
The ~40% reduction in LDL uptake resulting from exocyst disruption in WT cells is unlikely to be fully explained by an LDLR-independent effect.
“the ~40% reduction in LDL uptake resulting from exocyst disruption in WT cells is unlikely to be fully explained by an LDLR-independent effect”
Quote in the paperSubstantial claim - Interpretationreasoning
PCSK9-targeted cells are still susceptible to the activity of PCSK9 secreted by neighboring cells, preventing these mutants from developing alterations in LDLR abundance and LDL uptake.
“Therefore, PCSK9-targeted cells are still susceptible to the activity of PCSK9 secreted by neighboring cells, preventing these mutants from developing alterations in LDLR abundance and LDL uptake.”
Quote in the paperSubstantial claim- Rests on an assumption: PCSK9 induces LDLR degradation after its secretion.
- Study design and conductreasoning
Our screen may not uncover genes truly involved in LDL uptake if those genes also are either essential or confer a fitness advantage in culture, since gRNAs targeting those genes will be progressively depleted from the pooled population over the duration of the experiment.
“Fourth, our screen may not uncover genes truly involved in LDL uptake if those genes also are either essential or confer a fitness advantage in culture, since gRNAs targeting those genes will be progressively depleted from the pooled population over the duration of the experiment.”
Quote in the paperNot a substantial claim - Study design and conductreasoning
Our screen is unable to detect genes that perform redundant functions in LDL uptake, as compensation may prevent a significant phenotypic effect.
“Fifth, our screen is unable to detect genes that perform redundant functions in LDL uptake, as compensation may prevent a significant phenotypic effect.”
Quote in the paperSubstantial claim - Interpretationreasoning
Our findings are consistent with a model in which the exocyst is preferentially recruited to and promotes the recycling of LDLR-containing vesicles.
“Our findings are consistent with a model in which the exocyst is preferentially recruited to and promotes the recycling of LDLR-containing vesicles.”
Quote in the paperSubstantial claim - Interpretationown data
Orthogonal testing of our customized gRNA library provided us with initial insight into the mechanism of effect for each of our screen hits.
“Orthogonal testing of our customized gRNA library provided us with initial insight into the mechanism of effect for each of our screen hits.”
Quote in the paperSubstantial claim - Interpretationown data
The SREBP axis regulates additional mediators of LDL endocytosis beyond LDLR.
“suggesting that the SREBP axis regulates additional mediators of LDL endocytosis beyond LDLR”
Quote in the paperSubstantial claim - Interpretationreasoning
LDL uptake regulators may therefore still contribute to LDL uptake on a LDLR-deleted background if they also influence SCARB1 or other pathways mediating this residual LDL uptake.
“LDL uptake regulators may therefore still contribute to LDL uptake on a LDLR-deleted background if they also influence SCARB1 or other pathways mediating this residual LDL uptake.”
Quote in the paperSubstantial claim - Interpretationreasoning
Our findings provide further genetic support for a role of the exocyst in LDLR regulation by HuH7 cells.
“Our findings provide further genetic support for a role of the exocyst in LDLR regulation by HuH7 cells.”
Quote in the paperSubstantial claim - Interpretationreasoning
Generation of a secondary library facilitates additional assays providing further biologic insight into screen hits, as we were more readily able to query candidate genes under different selective pressures.
“Generation of a secondary library also facilitates additional assays providing further biologic insight into screen hits, as we were more readily able to query candidate genes under different selective pressures.”
Quote in the paperSubstantial claim
- Probably not a substantial claim. 29
- It may state two separate claims as one. 42
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationHuman genome-wide association studies have identified more than 250 loci associated with blood lipid levels.
“Human genome-wide association studies (GWAS) have also identified >250 loci associated with blood lipid levels[9–11].”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [9] CJ Willer, 2013, Discovery and refinement of loci associated with lipid levels, Nat Genet source
The Discussion reports that this study brought the total number of lipid-associated loci to 157, not more than 250. The Introduction and Results likewise report 157 loci, including 62 new ones. The sentence cites multiple sources, but this paper does not support the stated total. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured - cites [10] CS Paththinige, 2017, Genetic determinants of inherited susceptibility to hypercholesterolemia—a comprehensive literature review, Lipids Health Dis source
The section “Other loci associated with plasma lipid levels identified by GWAS” says that many GWAS have identified variants associated with serum lipid traits, supporting the general point. It does not establish the claimed count of more than 250 loci; another section reports over 190 variants in about 60 genes, which is a different measure. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [11] GM Peloso, 2018, Insights from population-based analyses of plasma lipids across the allele frequency spectrum, Curr Opin Genet Dev source
It may state two separate claims as one.
PremisecitationHuH7 cells are widely used as a model of hepatocyte LDL uptake and exhibit functional regulatory networks of cholesterol regulation and uptake, SREBP regulation, and statin sensitivity.
“HuH7 cells, originally derived from a well-differentiated human hepatocellular carcinoma [15], are widely used as a model of hepatocyte LDL uptake and exhibit functional regulatory networks of cholesterol regulation and uptake, SREBP regulation, and statin sensitivity[16].”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [15] H Nakabayashi, 1982, Growth of human hepatoma cells lines with differentiated functions in chemically defined medium, Cancer Res
- cites [16] P Blattmann, 2017, Systems Pharmacology Dissection of Cholesterol Regulation Reveals Determinants of Large Pharmacodynamic Variability between Cell Lines, Cell Syst source
The Results sections describe Huh7 as a well-established liver-derived model for drug response and metabolism, and show functional cholesterol regulation, SREBP2 responses, cholesterol uptake capacity, and statin sensitivity (pages not provided). They do not establish that Huh7 is widely used specifically as a model of hepatocyte LDL uptake. The Discussion cautions that Huh7 relies more on cholesterol synthesis than in vivo hepatocytes and that its predicted uptake behavior still needs experimental confirmation. (LLM, conf=0.85)
Experimental — accuracy not yet measured
Probably not a substantial claim.
PremisecitationDysregulation of cholesterol homeostasis is a key factor in the pathogenesis of atherosclerosis.
“A preponderance of evidence from epidemiology, human genetics, animal studies, and clinical trials have established that dysregulation of cholesterol homeostasis is a key factor in the pathogenesis of atherosclerosis[1].”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [1] BA Ference, 2017, Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel, Eur Heart J source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationSREBP signaling provides an additional level of regulation of genes involved in cholesterol uptake and synthesis, in which low cellular sterol levels lead to SCAP-mediated trafficking of SREBP proteins from the ER to the Golgi, where they are cleaved by MBTPS1 and MBTPS2 to release and activate their transcription factor domains.
“An additional level of regulation of the genes involved in cholesterol uptake and synthesis is provided by SREBP signaling, in which low cellular sterol levels lead to the SCAP-mediated trafficking of SREBP proteins from the ER to the Golgi, where they are cleaved by resident proteases (encoded by MBTPS1 and MBTPS2) to release and activate their transcription factor domains[7,8].”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [7] JD Horton, 2002, SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver, J Clin Invest source
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured - cites [8] MS Brown, 1997, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe exocyst is an octameric protein complex that was originally identified for its role in vesicular trafficking in budding yeast.
“an octameric protein complex that was originally identified for its role in vesicular trafficking in budding yeast[20,21]”
Quote in the paperNot a substantial claim1 of 2 sources checkedProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPrior investigations have implicated the retriever, CCC, and WASH complexes in LDLR recycling.
“Prior investigations have implicated the retriever, CCC, and WASH complexes in LDLR recycling[29,30]”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [29] P Bartuzi, 2016, CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL, Nat Commun source
The Discussion (page not provided) says CCC and WASH are required for LDLR endosomal sorting and directing LDLR to the plasma membrane, supporting their involvement in recycling. The paper does not identify the retriever complex as involved in LDLR recycling, so this source supports only two of the three named complexes. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [30] KE McNally, 2017, Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling, Nat Cell Biol source
The Results section describes retriever, CCC and WASH as components of an endosomal recycling pathway, and reports that the LDL receptor–related protein LRP1 depends on retriever for its cell-surface level. It does not establish that these complexes participate in recycling LDLR itself; the paper’s direct recycling experiments concern α5β1-integrin. (LLM, conf=0.85)
Experimental — accuracy not yet measured
It may state two separate claims as one.
PremisecitationClinical findings show that statins reduce LDL cholesterol and mortality in LDLR null patients with homozygous familial hypercholesterolemia.
“consistent with clinical findings that statins reduce LDL cholesterol and mortality in LDLR null patients with homozygous familial hypercholesterolemia[31]”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] M Cuchel, 2014, Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society, Eur Heart J source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe exocyst is an octameric protein complex involved in vesicular trafficking.
“Among the candidate LDL uptake modifiers identified in our primary CRISPR screen were multiple components of the exocyst, an octameric protein complex involved in vesicular trafficking[20].”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [20] B Wu, 2015, The Exocyst at a Glance, J Cell Sci source
It may state two separate claims as one.
PremisecitationSCARB1 encodes a scavenger receptor that binds a variety of ligands including LDL, has SREBP-binding sites in its promoter region, and is expressed in hepatocytes.
“SCARB1 encodes a scavenger receptor that binds a variety of ligands including LDL, has SREBP-binding sites in its promoter region, and is expressed in hepatocytes[32].”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [32] WJ Shen, 2018, Scavenger receptor B type 1: expression, molecular regulation, and cholesterol transport function, J Lipid Res source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationThe exocyst is recruited to vesicle membranes by small GTPases and mediates the tethering of these vesicles to target membranes.
“The exocyst is recruited to vesicle membranes by small GTPases and mediates the tethering of these vesicles to target membranes.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [20] B Wu, 2015, The Exocyst at a Glance, J Cell Sci source
The Introduction says activated Sec4, a small GTPase, might recruit the exocyst to secretory vesicles, and describes the exocyst as mediating vesicle tethering to the plasma membrane. But its vesicle-tethering discussion says direct evidence is missing. The claim states both mechanisms more definitively than the source does. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [21] P Novick, 1980, Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway, Cell source
Probably not a substantial claim.
Study design and conductcitationAssociation analysis for LDL cholesterol was performed using SAIGE for 388,629 individuals in the white British subset of UK Biobank.
“Association analysis for LDL cholesterol was performed using SAIGE[37] for 388,629 individuals in the white British subset of UK Biobank[38].”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [37] W Zhou, 2018, Efficiently controlling for case-control imbalance and sample relatedness in large-scale genetic association studies, Nat Genet source
- cites [38] C Sudlow, 2015, UK biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age, PLoS Med. source
It may state two separate claims as one.
Findingown dataThe secondary screen identified 45 negative regulators of HuH7 LDL uptake, with 18 of these exhibiting significant effects under both lipoprotein-rich and lipoprotein-depleted conditions.
“We also identified 45 negative regulators, with 18 of these exhibiting significant effects under both lipoprotein-rich and lipoprotein-depleted conditions (Fig 2D).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataDisruption of several genes resulted in decreased LDL uptake but enhanced transferrin uptake.
“Surprisingly, disruption of several genes resulted in decreased LDL uptake but enhanced transferrin uptake.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe positive control MYLIP, encoding the LDLR negative regulator IDOL, was ranked 7 and 138 among negative regulators under lipoprotein-rich and lipoprotein-depleted conditions but did not meet the FDR<5% threshold for genome-wide significance.
“The positive control MYLIP, encoding the LDLR negative regulator IDOL[18], was ranked 7 and 138 among negative regulators under lipoprotein-rich and lipoprotein-depleted conditions but did not meet the FDR<5% threshold for genome-wide significance.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [18] N Zelcer, 2009, LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor, Science source
It may state two separate claims as one.
Findingown dataOf the 163 identified genes, 12% (20/163) had a genome-wide significant GWAS result (p-value < 5x10-8) within the gene while 33% (54/163) had a significant result within 500 kb.
“Of these, 12% (20/163) had a genome-wide significant GWAS result (p-value < 5x10-8) within the gene while 33% (54/163) had a significant result within 500 kb.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataMany modifiers of LDL uptake identified in wild-type cells were also identified in LDLR-deleted cells, with 45/118 positive and 17/45 negative regulators exhibiting similar effects on LDL uptake in LDLR-deleted cells.
“Surprisingly, we found that many modifiers of LDL uptake identified in wild-type cells were also identified in LDLR-deleted cells, with 45/118 positive and 17/45 negative regulators exhibiting similar effects on LDL uptake in LDLR-deleted cells (Fig 4F–4I).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataEach internal control gene was identified with more significant enrichment and rose in the relative rankings, with LDLR and MYLIP becoming the top hits for reduced and enhanced LDL uptake, respectively, under both lipoprotein-rich and lipoprotein-depleted culture conditions.
“Each of these internal control genes was identified with more significant enrichment (Fig 2I) and rose in the relative rankings, with LDLR and MYLIP becoming the top hits for reduced and enhanced LDL uptake, respectively, both under lipoprotein-rich and lipoprotein-depleted culture conditions.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAs in HuH7 cells, LDL uptake in HepG2 cells was dependent on LDLR and modulated by targeting of known regulators including SCAP, MBTPS1, MBTPS2, and MYLIP.
“As in HuH7 cells, LDL uptake in HepG2 cells was dependent on LDLR and modulated by targeting of known regulators including SCAP, MBTPS1, MBTPS2, and MYLIP (Fig 6A and Fig 6D and S10 Table).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataUnder lipoprotein-rich conditions, the authors identified only 10 and 2 genes whose targeting was associated with reduced or increased LDL uptake in HepG2 cells, respectively, with 6/10 positive regulators and 1/2 negative regulators exhibiting similar effects in HuH7 cells.
“Under lipoprotein-rich conditions, we identified only 10 and 2 genes whose targeting was associated with reduced or increased LDL uptake in HepG2 cells, respectively (Fig 6A), with 6/10 positive regulators (Fig 6B) and 1/2 negative regulators (Fig 6C) exhibiting similar effects in HuH7 cells.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAs in the primary screen, genes identified under either lipoprotein-rich or lipoprotein-depleted conditions were much more likely to be identified under the other condition, with a high degree of correlation for the relative effect size under either condition.
“As in the primary screen, genes identified under either lipoprotein-rich or lipoprotein-depleted conditions were much more likely to be identified under the other condition (S5A and S5B Fig), with a high degree of correlation for the relative effect size under either condition (Fig 2F).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAmong the genes identified under lipoprotein-depleted conditions in HepG2 cells, 25/53 positive regulators and 2/5 negative regulators exhibited similar effects in HuH7 cells.
“Among these latter genes, 25/53 positive regulators (Fig 6E) and 2/5 negative regulators (Fig 6F) exhibited similar effects in HuH7 cells.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataPre-incubation of HuH7 cells with lipoprotein-depleted media resulted in an LDLR-dependent ~67% increase in LDL uptake, consistent with upregulation of LDLR expression via SREBP signaling.
“Pre-incubation of HuH7 cells with lipoprotein-depleted media resulted in an LDLR-dependent ~67% increase in LDL uptake, consistent with upregulation of LDLR expression via SREBP signaling (S1D Fig).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataThe top hit associated with both decreased surface and decreased total LDLR was LDLR itself, and the top hit for increased surface and increased total LDLR was MYLIP.
“As expected, the top hit associated with both decreased surface and decreased total LDLR was LDLR itself, and the top hit for increased surface and increased total LDLR was MYLIP.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataUsing an FDR cutoff of 5% in the secondary screen, the authors identified 118 positive regulators of HuH7 LDL uptake, with 66 of these exhibiting significant effects under both lipoprotein-rich and lipoprotein-depleted conditions.
“Using an FDR cutoff of 5% in our secondary screen, we identified 118 positive regulators of HuH7 LDL uptake (Fig 2A and 2B and S3 Table), with 66 of these exhibiting significant effects under both lipoprotein-rich and lipoprotein-depleted conditions (Fig 2C).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataPositive control genes LDLR, SCAP, and MBTPS1 were again positively identified in the secondary screen, while MBTPS2 and MYLIP were readily detected as positive hits in the secondary screen.
“Positive control genes LDLR, SCAP, and MBTPS1 were again positively identified in the secondary screen, while MBTPS2 and MYLIP (negative in the primary screen) were readily detected as positive hits in the secondary screen.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataTargeted genes demonstrated a high degree of correlation between surface and total LDLR staining, with no genes exhibiting significant effects on surface and total LDLR staining in opposite directions.
“Targeted genes demonstrated a high degree of correlation between surface and total LDLR staining (Fig 5F), with no genes exhibiting significant effects on surface and total LDLR staining in opposite directions.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataMBTPS2 was not identified among the primary screen hits, ranking 343 and 58 under lipoprotein-rich and lipoprotein-depleted conditions with FDR>5% for both.
“Among these candidates were known regulators of LDL uptake including LDLR, SCAP, and MBTPS1, though MBTPS2 was not identified among the screen hits (ranking 343 and 58 under lipoprotein-rich and lipoprotein-depleted conditions, with FDR>5% for both).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataDisruption of most LDL uptake regulators did not cause a similar reduction in transferrin uptake and for some genes instead caused opposing effects.
“Disruption of most LDL uptake regulators did not cause a similar reduction in transferrin uptake and for some genes instead caused opposing effects.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
FindingreasoningWe observed significantly improved signal-to-noise ratio in our secondary screen, with more significant enrichment for positive hits, improved detection of internal positive controls, and no identification of negative control nontargeting genes.
“Reflecting these technical advantages, we observed significantly improved signal-to-noise ratio in our secondary screen, with more significant enrichment for positive hits, improved detection of internal positive controls, and no identification of negative control nontargeting genes.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataLDLR and MYLIP were the top hits for positive and negative regulation of cell surface and total LDLR abundance in HuH7 cells.
“as well as for positive and negative regulation of cell surface and total LDLR abundance in HuH7 cells.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataMutant EXOC4 and EXOC8 clones each exhibited a ~40% decrease in LDL uptake and a ~2-3 fold increase in transferrin uptake, both of which were rescued by expression of the corresponding cDNA.
“Mutant EXOC4 and EXOC8 clones each exhibited a ~40% decrease in LDL uptake and a ~2–3 fold increase in transferrin uptake, both of which were rescued by expression of the corresponding cDNA (Fig 7D and 7E).”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper may give no reason why it holds.
Findingnone givenThe immortalized hepatoma cell system recapitulates the LDLR-dependent, SREBP-responsive nature of cellular LDL uptake.
“While it is reassuring that this system recapitulates the LDLR-dependent, SREBP-responsive nature of cellular LDL uptake”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataWe identified a list of high-confidence genetic modifiers of HuH7 cell LDL uptake, with supporting evidence for their specificity, mechanism of action, and generalizability.
“In summary, we identified a list of high-confidence genetic modifiers of HuH7 cell LDL uptake, with supporting evidence for their specificity, mechanism of action, and generalizability.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataLDLR and MYLIP were the top hits for positive and negative regulation of LDL uptake in both HuH7 and HepG2 cells, under both lipoprotein-rich and lipoprotein-depleted conditions.
“with LDLR and MYLIP representing the top hits for positive and negative regulation of LDL uptake in both HuH7 and HepG2 cells, under both lipoprotein-rich and lipoprotein-depleted conditions”
Quote in the paperSubstantial claimProbably not a substantial claim.
Study design and conductnone givenA total of 3 independent biologic replicates were performed for each screen.
“A total of 3 independent biologic replicates were performed for each screen.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown dataThe HuH7 model system recapitulates the LDLR-dependence and SREBP-responsiveness of cellular LDL uptake and is a suitable platform for genome-wide screening.
“These results suggest that this model system recapitulates the LDLR-dependence and SREBP-responsiveness of cellular LDL uptake and is a suitable platform for genome-wide screening.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Study design and conductown dataMassively parallel sequencing of the plasmid pool of the custom library confirmed the presence of 99.97% of gRNA sequences inserted into the CRISPR plasmid backbone, with minimal library skewing.
“Massively parallel sequencing of the plasmid pool of this library confirmed the presence of 99.97% of gRNA sequences inserted into the CRISPR plasmid backbone, (S3A Fig) with minimal library skewing (S3B Fig).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Study design and conductown dataThe LDL uptake assay conditions were in the linear range of detection with respect to both LDL dose and duration of incubation.
“These conditions were in the linear range of detection with respect to both LDL dose and duration of incubation (S1B and S1C Fig).”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationLDL cholesterol levels are tightly controlled through the complex interplay between intestinal absorption of dietary cholesterol, de novo cholesterol biosynthesis, efflux of cholesterol from peripheral tissues, and cellular uptake of lipoproteins.
“LDL cholesterol levels are tightly controlled through the complex interplay between intestinal absorption of dietary cholesterol, de novo cholesterol biosynthesis, efflux of cholesterol from peripheral tissues, and cellular uptake of lipoproteins[2].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [2] J Luo, 2019, Mechanisms and regulation of cholesterol homeostasis, Nat Rev Mol Cell Biol source
It may state two separate claims as one.
Premisenone givenFor the majority of GWAS associations, the causal link to a specific gene and the mechanism for the genotype-phenotype correlation remains unknown.
“For the majority of GWAS associations, the causal link to a specific gene and the mechanism for the genotype-phenotype correlation remains unknown.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
InterpretationreasoningMore genes were identified with FDR<5% in the secondary screen, suggesting an increased power of detection.
“More genes were identified with FDR<5%, suggesting an increased power of detection.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe authors previously performed a genome-wide CRISPR screen for the molecular determinants of PCSK9 secretion, leading to identification of SURF4 as a cargo receptor that recruits PCSK9 into the secretory pathway.
“We previously performed a genome-wide CRISPR screen for the molecular determinants of PCSK9 secretion, leading to our identification of SURF4 as a cargo receptor that recruits PCSK9 into the secretory pathway[14].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] BT Emmer, 2018, The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9, Elife source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationThe importance of LDL uptake to human cholesterol regulation and cardiovascular disease is highlighted by monogenic causes of familial hypercholesterolemia affecting this pathway, including mutations in LDLR, apolipoprotein B, PCSK9, or LDLRAP1.
“The importance of LDL uptake to human cholesterol regulation and cardiovascular disease is highlighted by the monogenic causes of familial hypercholesterolemia that affect this pathway[6], including mutations in the genes encoding for LDLR itself, its ligand apolipoprotein B, its negative regulator PCSK9, or its endocytic adapter LDLRAP1.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [6] DJ Rader, 2003, Monogenic hypercholesterolemia: new insights in pathogenesis and treatment, J Clin Invest source
Checked before — its earlier verdict is held back until a new check runs.
It may state two separate claims as one.
Interpretationown dataThe identification of several positive controls and the concordance of hits across screening conditions suggested that the primary screen was successful, though limited by background noise at genome scale.
“The identification of several positive controls and the concordance of our hits across screening conditions suggested that our primary screen was successful, though limited by background noise at genome scale.”
Quote in the paperSubstantial claimProbably not a substantial claim. It may state two separate claims as one.
Interpretationnone givenThe molecular basis for residual LDL uptake in LDLR-deleted cells is not well understood, but may be mediated by alternative receptors for LDL.
“The molecular basis for residual LDL uptake in LDLR-deleted cells is not well understood, but may be mediated by alternative receptors for LDL.”
Quote in the paperSubstantial claimProbably not a substantial claim.
Study design and conductnone givenCell numbers were maintained above a minimum depth of coverage of 500X relative to the customized gRNA library throughout the screen until the time of sorting.
“Cell numbers were maintained above a minimum depth of coverage of 500X relative to the customized gRNA library throughout the screen until the time of sorting.”
Quote in the paperSubstantial claimProbably not a substantial claim. It may state two separate claims as one.
Study design and conductnone givenSingle cell clones were analyzed by Sanger sequencing and TIDE deconvolution of PCR amplicons at the CRISPR target site, and by immunoblotting with antibodies against LDLR, EXOC4, EXOC8, and β-actin.
“Single cell clones were analyzed by Sanger sequencing and TIDE deconvolution of PCR amplicons at the CRISPR target site, and by immunoblotting with antibodies against LDLR, EXOC4, EXOC8, and β-actin.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
InterpretationreasoningIt is likely that among our list of hits are a subset of false positives, and likewise that among our genes which did not pass validation are a number of false negatives.
“It is likely that among our list of hits are a subset of false positives, and likewise that among our genes which did not pass validation are a number of false negatives.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenThree replicates were performed for each screen.
“Three replicates were performed for each screen.”
Quote in the paperNot a substantial claimProbably not a substantial claim. It may state two separate claims as one.
Study design and conductnone givenOur screen was performed in immortalized hepatoma cells, removed from the in vivo environment and evaluated in two-dimensional cell culture.
“First, our screen was performed in immortalized hepatoma cells, removed from the in vivo environment and evaluated in two-dimensional cell culture.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Study design and conductown dataImmunoblotting confirmed a loss of protein in each mutant cell line with restoration by ectopic expression of the CRISPR-resistant cDNA.
“Immunoblotting confirmed a loss of protein in each mutant cell line with restoration by ectopic expression of the CRISPR-resistant cDNA (Fig 7C).”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductcitationGenotyping was performed by Sanger sequencing of PCR amplicons of genomic target sites with individual alleles deconvoluted by TIDE analysis of chromatograms.
“Genotyping was performed by Sanger sequencing of PCR amplicons of genomic target sites with individual alleles deconvoluted by TIDE analysis of chromatograms[33].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [33] EK Brinkman, 2014, Easy quantitative assessment of genome editing by sequence trace decomposition, Nucleic Acids Res source
Probably not a substantial claim.
PremisecitationA prior study found that intracellular injection of an antibody against Sec8 (encoded by EXOC4) into polarized MDCK cells disrupted LDLR trafficking.
“A prior study found that intracellular injection of an antibody against Sec8 (encoded by EXOC4) into polarized MDCK cells disrupted LDLR trafficking[22].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [22] KK Grindstaff, 1998, Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells, Cell source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Interpretationnone givenThe molecular mechanism by which the exocyst promotes LDL uptake is unclear but seems likely to affect the kinetics of LDLR recycling.
“The molecular mechanism by which the exocyst promotes LDL uptake is unclear but seems likely to affect the kinetics of LDLR recycling.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationLDL and transferrin initially enter into common endosomes via clathrin-mediated endocytosis but then quickly segregate.
“LDL and transferrin initially enter into common endosomes via clathrin-mediated endocytosis but then quickly segregate[26–28].”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [26] PA Keyel, 2006, A single common portal for clathrin-mediated endocytosis of distinct cargo governed by cargo-selective adaptors, Mol Biol Cell source
- cites [27] KW Dunn, 1989, Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome, J Cell Biol source
Checked before — its earlier verdict is held back until a new check runs.
- cites [28] RN Ghosh, 1994, Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy, J Cell Sci source
It may state two separate claims as one.
Interpretationown dataOur findings provide further support for the involvement of these genes in human cholesterol regulation and suggest a molecular mechanism for their involvement in human lipid traits.
“Our findings provide further support for the involvement of these genes in human cholesterol regulation and suggest a molecular mechanism for their involvement in human lipid traits.”
Quote in the paperSubstantial claimProbably not a substantial claim.
Study design and conductnone givenTotal cell numbers were maintained above 25 million cells (representing 200X coverage of the gRNA library) throughout the entirety of the screen.
“Total cell numbers were maintained above 25 million cells (representing 200X coverage of the gRNA library) throughout the entirety of the screen.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductcitationLibrary diversity was assessed with a Illumina MiSeq run of gRNA amplicons prepared as previously described.
“Library diversity was assessed with a Illumina MiSeq run of gRNA amplicons prepared as previously described[14].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] BT Emmer, 2018, The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9, Elife source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationReceptor recycling from endosomes is a complex process that involves distinct pathways involving different adaptors, protein sorting machinery, and membrane composition.
“Receptor recycling from endosomes is a complex process that involves distinct pathways involving different adaptors, protein sorting machinery, and membrane composition[23–25].”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [23] GMI Redpath, 2020, Membrane Heterogeneity Controls Cellular Endocytic Trafficking, Front Cell Dev Biol source
Checked before — its earlier verdict is held back until a new check runs.
- cites [24] N Naslavsky, 2018, The enigmatic endosome—sorting the ins and outs of endocytic trafficking, J Cell Sci source
Checked before — its earlier verdict is held back until a new check runs.
- cites [25] S Weeratunga, 2020, Recognising the signals for endosomal trafficking, Curr Opin Cell Biol source
Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Interpretationown dataThese findings highlight the power of genome-scale CRISPR screening and offer new avenues for understanding the molecular determinants of cellular LDL uptake.
“These findings highlight the power of genome-scale CRISPR screening and offer new avenues for understanding the molecular determinants of cellular LDL uptake.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Interpretationnone givenThe basis for the discrepancy between LDL uptake effects and LDLR levels is unclear but may be related either to screen hits influencing LDLR kinetics or function rather than steady state levels, or to compensatory effects in mutant cells that upregulate LDLR expression in response to defective LDL uptake.
“The basis for this discrepancy is unclear but may be related either to screen hits influencing LDLR kinetics or function rather than steady state levels, or to compensatory effects in mutant cells that upregulate LDLR expression in response to defective LDL uptake.”
Quote in the paperNot a substantial claimProbably not a substantial claim. It may be too vague to check against evidence.
Interpretationown dataOur findings highlight the value of following up candidate genes from a primary genome-wide CRISPR screen with a customized gRNA library.
“Our findings highlight the value of following up candidate genes from a primary genome-wide CRISPR screen with a customized gRNA library.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
PCSK9 induces LDLR degradation after its secretion.
“PCSK9 induces LDLR degradation after its secretion.”
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?
Experimental evidence has recapitulated key modulators of cellular LDL uptake, as well as posttranscriptional and posttranslational regulators of LDLR.
“Moreover, experimental evidence has recapitulated key modulators of cellular LDL uptake, as well as posttranscriptional and posttranslational regulators of LDLR79–82.”
From ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver (2025) · cites this paper as [79]
The Results sections on the primary and secondary CRISPR screens report recovery of known LDL-uptake regulators, including LDLR, SCAP, MBTPS1, MBTPS2, and MYLIP. The LDLR-staining Results and Discussion also support regulation of LDLR protein levels and trafficking, but the passages do not establish that the screen recapitulated posttranscriptional regulators of LDLR. Page numbers are unavailable in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measuredIndividual cells with disrupted PCSK9 will remain sensitive to the extracellular PCSK9 secreted by neighboring cells, precluding their development of a LDLR-mediated phenotype in this study and in prior pooled CRISPR screens.
“For example, individual cells with disrupted PCSK9 will remain sensitive to the extracellular PCSK9 secreted by neighboring cells, precluding their development of a LDLR-mediated phenotype in this study and in prior pooled CRISPR screens for LDL uptake or LDLR abundance56,80,93.”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
The Discussion (page not provided) says that PCSK9-targeted cells remain susceptible to PCSK9 secreted by neighboring cells, preventing changes in LDLR abundance and LDL uptake in this screen. It does not establish that the same outcome occurred in the prior pooled CRISPR screens mentioned in the citing sentence. (LLM, conf=0.84)
Experimental — accuracy not yet measuredA prior genome-scale CRISPR screen successfully identified regulators of cellular LDL endocytosis.
“We previously applied a genome-scale CRISPR screen to successfully identify regulators of cellular LDL endocytosis56.”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
The smaller genetic effect on Lp(a) is consistent with our lipoprotein uptake studies and in vitro binding studies.
“This finding is consistent with our lipoprotein uptake studies that revealed a less pronounced defect in LDLR-disrupted cells for Lp(a) uptake (~61% reduction) compared to LDL uptake (~87% reduction56), and with our in vitro binding studies showing a reduced affinity for LDLR binding to Lp(a) relative to LDL.”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
RAB10-targeted HuH7 cells exhibit increased accumulation of fluorescently labeled transferrin relative to control cells.
“RAB10-targeted cells exhibited decreased uptake of fluorescently labeled LDL and increased accumulation of fluorescently labeled transferrin relative to control cells treated with a nontargeting gRNA (Fig. 1B, C).”
From The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes (2022) · cites this paper as [13]
The Results section on transferrin uptake reports that disrupting several genes decreased LDL uptake while enhancing transferrin uptake in HuH7 cells, but it does not identify RAB10 among them. Searches for RAB10 and RAB10-targeted cells found no passage establishing increased transferrin accumulation relative to controls. (LLM, conf=0.73)
Experimental — accuracy not yet measured
The previously reported CRISPR screen for modifiers of LDL uptake identified RABIF, a guanine nucleotide exchange factor that stimulates GDP release from various Rab GTPases including RAB10, and which has also been shown to stabilize RAB10.
“The previously reported CRISPR screen for modifiers of LDL uptake (13) also identified RABIF (Rab interacting factor), a guanine nucleotide exchange factor that stimulates GDP release from various Rab GTPases including RAB10, and which has also been shown to stabilize RAB10 (46).”
From The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes (2022) · cites this paper as [13]
The Results sections describe the CRISPR screen and its LDL-uptake hits, but searches for “RABIF” and “Rab interacting factor” found no passage identifying RABIF as a hit in our copy. The sentence cites this paper for that identification; it separately cites reference 46 for RABIF’s activity and RAB10 stabilization. (LLM, conf=0.72)
Experimental — accuracy not yet measuredRAB10 is a positive regulator of LDL uptake in HuH7 cells in a genome-wide CRISPR screen.
“This screen identified RAB10, a small GTPase known to mediate trafficking of vesicles between intracellular compartments, as a key regulator of LDL uptake.”
From The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes (2022) · cites this paper as [13]
RAB10-targeted HuH7 cells exhibit decreased uptake of fluorescently labeled LDL relative to control cells.
“RAB10-targeted cells exhibited decreased uptake of fluorescently labeled LDL and increased accumulation of fluorescently labeled transferrin relative to control cells treated with a nontargeting gRNA (Fig. 1B, C).”
From The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes (2022) · cites this paper as [13]
Our prior screen of LDL uptake modifiers identified several exocyst components (EXOC1, EXOC2, EXOC3, EXOC4, EXOC7, EXOC8) that phenocopied RAB10, with depletion of either protein resulting in decreased LDL uptake and increased transferrin accumulation.
“Our prior screen of LDL uptake modifiers likewise identified several exocyst components including EXOC1, EXOC2, EXOC3, EXOC4, EXOC7, and EXOC8 that phenocopied RAB10, with depletion of either protein resulting in decreased LDL uptake and increased transferrin accumulation.”
From The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes (2022) · cites this paper as [13]
The prior CRISPR screen detected expected genes encoding the LDL receptor and its canonical transcriptional and posttranscriptional regulators.
“We readily detected expected genes encoding the LDL receptor and its canonical transcriptional and posttranscriptional regulators.”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
The prior CRISPR screen detected novel regulators of LDL uptake that were reproducible across independent replicates and during follow-up testing.
“We also detected novel regulators of LDL uptake that were reproducible across independent replicates and during follow up testing56,57.”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
The screen used the genome-wide GeCKOv2 CRISPR knockout library.
“we next scaled our cellular Lp(a) uptake assay to query the same GeCKOv2 genome-wide CRISPR knockout library59 we tested in our prior screen of LDL uptake56”
From Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening (2025) · cites this paper as [56]
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Self-citation
Metadata
- Authors
- Emmer, B. T., Sherman, E. J., Lascuna, P. J., Graham, S. E., Willer, C. J., & Ginsburg, D.
- Affiliations
- University of Michigan (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Ginsburg, David
- Venue
- PLOS Genetics
- Publisher
- Public Library of Science (PLoS)
- Published
- 2021-01-29
- Volume
- 17
- Issue
- 1
- Pages
- e1009285
- ISSN
- 1553-7390 (ISSN-L)
- 1553-7404
- PMC
- PMC7875399
- OpenAlex
- W3125401634
- Semantic Scholar
- 5d39ad23a217163c346cb0f4f20f86c5fbbef4d6
- Cited by
- 32
- References
- 46
- Citations by year
- 2026: 7 · 2025: 7 · 2024: 1 · 2023: 8 · 2022: 5 · 2021: 4
- FWCI
- 1.82
- Type
- journal article
- Language
- en
- Update notices
- new_version (2021-02-10)
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS Genetics (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Heart, Lung, and Blood Institute (R35-HL135793T, K08-HL148552)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
- Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipoproteins and Cardiovascular Health
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- LDL receptor, LDL uptake, hypercholesterolemia, cholesterol homeostasis, transferrin endocytosis, genetic epistasis, Huh7 cells, atherosclerotic cardiovascular disease
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cholesterol, Endocytosis, Gene Expression Regulation, Humans, Hypercholesterolemia, Lipoproteins, LDL, Receptors, LDL, Genome, Human, Hepatocytes, Atherosclerosis, Hep G2 Cells, 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 2026-11-09, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-11-09 (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.
What counts as a claim ›How we measure confidence ›How we check this ›
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