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CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Open accessAbstract
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
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In the paper
“Here we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells with the site of delivey determined solely by a co-expressed short guide (sg)RNA.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > Abstract
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In the paper
“In this experiment we found that we can simultaneously knock down endogenous expression of CD71 and CXCR4 (Figure 3E).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The fluorescent antibodies used to stain CD71 and CXCR4 are specific for those proteins.
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
“Without dCas9, rtTA strongly activated the reporter, but this induction can be effectively blocked by dCas9 (115 fold repression, Figure 4B).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our data show CD71 and CXCR4 can be efficiently and stably knocked down (Figure 3C–D), providing evidence that CRISPRi can be used like RNAi to suppress endogenous gene expression.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The fluorescent antibodies used to stain CD71 and CXCR4 are specific for those proteins.
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
“Interestingly, a strong correlation exists between the degree of knockdown for dCas9 and dCas9-KRAB for endogenous genes”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The fluorescent antibodies used to stain CD71 and CXCR4 are specific for those proteins.
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
“Furthermore, we observed few changes in gene expression in the negative control sample, suggesting that the negative control sgRNA triggers minimal off-target effects (Figure 2 and Figure S3A).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi-mediated gene knockdown is highly specific to the target gene
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results revealed that both dCas9-VP64 and dCas9-p65AD can effectively activate reporter gene expression, suggesting that dCas9 can serve as a generic and modular platform for different types of transcriptional control including both repression and activation (Figure 1E).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We found that we can robustly silence GFP expression in HEK293 cells when both the RNA and protein components of CRISPRi are stably expressed in human cells (Figure 1D).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Interestingly, we also observed that blocking the AP1 enhancer caused a stronger silencing effect than blocking the SP1 enhancer within the SV40 promoter (Figure 4A)”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results also revealed a linear relationship between the level of expression from the sgRNA vector and the level of GFP remaining within a cell (Figure S2A).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Strength:plain· not yet calibratedCausal:correlational· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Finally, expression of dCas9-KRAB resulted in minimal changes in gene expression (Figure S3C).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi-mediated gene knockdown is highly specific to the target gene
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesNone of the sources cited for this claim have been checked yet.
In the paper
“The observation that the sgRNA is a limiting factor for CRISPRi function suggests that improvements to RNA stability or loading into dCas9 could improve both CRISPRi and Cas9 mediated genome editing in human cells (Jinek et al., 2013).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > Future optimization for CRISPRi technology
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“we observed robust gene knockdown of both reporter and endogenous genes (5~15-fold repression in human and 50-fold in yeast).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our data here describes a dramatic improvement from our previously published work on CRISPRi in human cells and argues that CRISPRi represents an alternate strategy to RNAi for repressing gene expression in mammalian cells.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Load-bearingStrength:boosted· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“we observed robust gene knockdown of both reporter and endogenous genes (5~15-fold repression in human and 50-fold in yeast).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesThe 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
“While we are able to target dCas9 to both endogenous and reporter genes, not all sgRNA targeting constructs work efficiently.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > Future optimization for CRISPRi technology
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“as we show that promoters and in theory introns can also be targeted.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our RNA-seq results show that CRISPRi is highly specific with minimal off-target effects for two distinct sgRNA sequences.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“suggesting that the dCas9-KRAB fusion protein might recruit chromatin modifying complexes to increase the potency of CRISPRi silencing.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIn the paper
“dCas9 fusion proteins can functionally mimic multiple native protein interactions, including the KRAB domain of Kox1, VP16 and p65AD (Groner et al., 2010).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > dCas9 provides a general platform for targeting proteins to DNA
What the sources say
- Not checked yetcites Groner et al., 2010 source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Here, we show that dCas9 can be used as a modular RNA-guided platform to recruit different protein effectors to DNA in a highly specific manner in human cells and the budding yeast Saccharomyces cerevisiae.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > INTRODUCTION
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“providing evidence that CRISPRi can be used like RNAi to suppress endogenous gene expression.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“implying that CRISPRi can be used as a tool for perturbing and mapping regulatory functions of DNA elements.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Taken together with the present study, these findings suggest that CRISPRi will be a general method for efficiently and specifically regulating transcription in many eukaryotes.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi is a general method for silencing transcription of endogenous genes in eukaryotes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The modularity of the dCas9 system provides a new approach for studying transcription, epigenetic regulation and both DNA replication and repair.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > dCas9 provides a general platform for targeting proteins to DNA
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“suggesting that sgRNA binding to DNA or local chromatin structure may be limiting factors which dictate gene knockdown (Figure S4A).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This suggests that dCas9 can sterically compete with transcription factors which have tight binding affinity for DNA elements”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results provide evidence that dCas9 can target enhancers, introns, and other noncoding elements to map the regulatory functions of these elements on transcription.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > Mapping the function of enhancers and chromosome topology with dCas9
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Furthermore, because CRISPRi acts at the DNA level, one can target transcripts such as non-coding RNAs, microRNAs, antisense transcripts, nuclear localized RNAs, and polymerase III transcripts.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: CRISPRi acts at the DNA level.
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 is comparable to the efficiency of existing gene knockdown techniques such as RNAi and TALE proteins.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Finally, CRISPRi possesses a much larger targetable sequence space, as we show that promoters and in theory introns can also be targeted.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our data here describes a dramatic improvement from our previously published work on CRISPRi in human cells and argues that CRISPRi represents an alternate strategy to RNAi for repressing gene expression in mammalian cells.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our data demonstrate how dCas9 can be used to target a protein to a specific region of DNA.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > dCas9 provides a general platform for targeting proteins to DNA
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“While we do not yet know what fraction of genes can be silenced by a dCas9 fusion protein, a significant portion of the genome should be targetable.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Load-bearingStrength:hedged· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“suggesting that dCas9 can serve as a generic and modular platform for different types of transcriptional control including both repression and activation (Figure 1E).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIn the paper
“By contrast, genome editing by the catalytically active Cas9 nuclease can be accompanied by off-target genomic alterations which occur at loci where near cognate DNA seed matches exist (Fu et al., 2013).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
What the sources say
- Not checked yetcites Fu et al., 2013
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes
- It may state two separate claims as one. 14
- Probably not a substantial claim. 7
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We found that cells expressing the dCas9-KRAB fusion protein show a 5-fold decrease in GFP signal while cells expressing dCas9 alone, dCas9-CS or dCas9-WRPW show a 2-fold decrease in GFP signal”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We also found no evidence for significant on- or off-target effects due to expression of either sgGAL4 or sgGFP-NT1 alone (Figure S3B).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi-mediated gene knockdown is highly specific to the target gene
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We found that while dCas9 alone had no effect on transcription, we efficiently repressed GFP expression with dCas9-KRAB (Figure 4A).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We observed that 6 out of 9 sgRNAs targeting GFP knocked down GFP expression by at least 75% with a ~15-fold repression for the best sgRNA (NT1) (Table S1).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We found that stable dCas9-KRAB expression is sufficient to silence GFP with substantial knockdown 3 days following transfection (Figure S1A–B) and strong silencing 6 days following transfection (Figure 1C).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results show that CRISPRi is highly specific as GFP is the only gene that is significantly suppressed by the GFP-targeting sgRNA (Figure 2 and Figure S3A). If we average the data from two independent biological replicates, no gene other than GFP changes by more than 1.5 fold.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi-mediated gene knockdown is highly specific to the target gene
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“With dCas9 alone, we observed 18-fold repression, which is increased to 53-fold with addition of the Mxi1 domain (Figure 3F).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi is a general method for silencing transcription of endogenous genes in eukaryotes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We sequenced the GFP reporter locus in these cells to confirm that dCas9 is completely nucleolytically inactive. Here, GFP retained a wild type sequence with no detectable indels in GFP knockdown cells.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In this experiment we found that both dCas9 and dCas9-KRAB proteins can knock down endogenous expression of CD71 and CXCR4 (Figure 3A–B), with 3 out of 10 sgRNAs for each gene showing significant repression (60–80% repression).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi can silence endogenous human genes
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
- Rests on an assumption: The fluorescent antibodies used to stain CD71 and CXCR4 are specific for those proteins.
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 we are able to target dCas9 to both endogenous and reporter genes, not all sgRNA targeting constructs work efficiently.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > Future optimization for CRISPRi technology
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“arguing that the design of the fusion protein or fusion partner is an important parameter for future optimization efforts.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > dCas9 fusion proteins can efficiently activate or silence transcription
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
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 suggests that while dCas9 may not be able to directly block the initiation of transcription, dCas9 fusion proteins can silence proximal regulatory elements within a promoter by recruiting chromatin modifiers.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > RESULTS > CRISPRi as a tool for mapping and perturbing regulatory elements
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“The specificity of CRISPRi remains to be determined across a broad range of sgRNA sequences.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“In human cells, previous work has demonstrated that local chromatin context is a crucial determinant of KRAB mediated silencing.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
What the sources say
- Not checked yetcites Groner et al., 2010 source
Strength:boosted· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
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
“Nonetheless, it may be substantially easier to specifically modulate transcription as only a small fraction of the human genome is transcribed or required for controlling transcription thus binding of catalytically inactive dCas9, and it is derivatives, at near cognate sites will often not impact transcription.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
- Rests on an assumption: Only a small fraction of the human genome is transcribed or required for controlling transcription.
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
“As an exogenous system, CRISPRi does not compete with endogenous machinery such as microRNA expression or function.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✗ Based: failsProbably not a substantial claim. The paper may give no 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
“as we show that promoters and in theory introns can also be targeted.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi provides an alternative strategy to RNAi for gene regulation
Strength:hedged· not yet calibratedCausal:conditional· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✗ Based: failsProbably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
In the paper
“For example, recruitment of the KRAB domain to adjacent genomic regions can have no effect or can completely silence transcription (Groner et al., 2010).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
What the sources say
- Not checked yetcites Groner et al., 2010 source
Strength:plain· not yet calibratedCausal:conditional· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes? Load-bearing: unclear✓ Based: passesIt may state two separate claims as one.
In the paper
“Nonetheless, an exon trapping strategy in which Tet binding sites were randomly inserted in the genome allowing recruitment of a Tetracycline repressor-KRAB fusion protein revealed that ~80% of genes tested can be silenced by KRAB-mediated recruitment of Kap1 and HP1 proteins (Groner et al., 2010).”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > DISCUSSION > CRISPRi efficiently and specifically represses transcription in human cells
What the sources say
- Not checked yetcites Groner et al., 2010 source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Triplicate cultures were measured for each experiment and their standard deviation was indicated as the error bar.”
§ CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes > METHODS > Strain construction and fluorescence measurements for CRISPRi in yeast
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
The paper relies on this without support.
The fluorescent antibodies used to stain CD71 and CXCR4 are specific for those proteins.
“We designed sgRNAs to target genes encoding cell surface transmembrane proteins, enabling us to use flow cytometry with directly conjugated fluorescent antibodies to quantify gene expression at the single-cell level.”
The paper relies on this without support.
Only a small fraction of the human genome is transcribed or required for controlling transcription.
“only a small fraction of the human genome is transcribed or required for controlling transcription”
The paper relies on this without support.
CRISPRi acts at the DNA level.
“because CRISPRi acts at the DNA level”
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?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“For example, CRISPRi (interference)23–25 and CRISPRa (activation)26–30 technologies utilize catalytically inactive Cas9 (dCas9) fused to either a transcriptional repressor (e.g., KRAB, Krüppel associated box) or a transcriptional activator (e.g., VPR, VP64-p65-Rta).”
From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [23]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“such as fusing dCas9 to direct or indirect transcription activators (such as VP64), to increase the expression of a specific DNA sequence”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [130]
The cited source says what the claim attributes to it.
In the citing paper
“or transcription repressors (such as KRAB), to increase the efficiency of dCas9-mediated transcription inhibition [130,131].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [130]
The check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“The National Institute of Standards and Technology (NIST) manages a scientific consortium aimed at measuring and standardizing such outcomes as genome editing technology advances 100 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [100]
Experimental — accuracy not yet measuredIn the citing paper
“Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a naturally occurring bacterial immune system that uses a novel nuclease system to protect bacteria from phage infection (6-9), and it has recently been harnessed for a variety of genome-engineering applications (10)(11)(12)(13)(14)(15)(16)(17).”
From RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection (2014) · cites this paper as [15]
Citations
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Self-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · cited 2×
“Recently, several groups have shown that a modified type II CRISPR (Clustered Regularly Interspaced Palindromic Repeats) system can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in a variety of organisms (Cho et al., 2013; Cong et al., 2013; Dicarlo et al., 2013; Gratz et al., 2013…”
BackgroundEnzymatic assembly of DNA molecules up to several hundred kilobases
Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009) · Nature Methods · doi:10.1038/nmeth.1318 · Cited by 10,088
RNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210 · cited 4×
“However, the remarkable success in using RNA-guided Cas9 to enable DNA cutting for many genes in distinct organisms argues that Cas9 can bind to target DNA sites in a broad range of genes or regulatory regions (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jiang et…”
BackgroundInfluentialRepurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016 · cited 3×
“dCas9 Fusion Proteins Can Efficiently Activate or Silence Transcription We have shown recently that CRISPRi can decrease gene expression in human cells (Qi et al., 2013).”
BackgroundInfluentialSelf-citationTight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Gossen, M., & Bujard, H. (1992) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.89.12.5547 · Cited by 4,943
Global analysis of protein localization in budding yeast
Huh, W.-K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S., & O'Shea, E. K. (2003) · Nature · doi:10.1038/nature02026 · Cited by 4,248
Self-citationWang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013) · Cell · doi:10.1016/j.cell.2013.04.025 · Cited by 3,495 · cited 2×
“…distinct organisms argues that Cas9 can bind to target DNA sites in a broad range of genes or regulatory regions (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jiang et al., 2013; Jinek et al., 2012, 2013; Mali et al., 2013; Wang et al., 2013).”
BackgroundHigh-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937
Flagged first, then by citations
Cited by
Sorted by citations
Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133
The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062
“Specifically, fusion of dCas9 to effector domains including VP64 or KRAB allowed stable and efficient transcriptional activation or repression, respectively, in human and yeast cells (129).”
Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762
“This CRISPR-based interference, or CRISPRi, works efficiently in prokaryotic genomes but is less effective in eukaryotic cells (Gilbert et al., 2013).”
InfluentialGenome-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
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153
Metadata
- Authors
- 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., Lim, W. A., Weissman, J. S., & Qi, L. S.
- Affiliations
- QB3 (US)
- Howard Hughes Medical Institute (US)
- Peking University (CN)
- Corresponding author
- Weissman, Jonathan S.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2013-07
- Volume
- 154
- Issue
- 2
- Pages
- 442-451
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC3770145
- OpenAlex
- W2101749257
- Cited by
- 3,696
- References
- 46
- Citations by year
- 2026: 195 · 2025: 260 · 2024: 298 · 2023: 286 · 2022: 316 · 2021: 374 · 2020: 387 · 2019: 332 · 2018: 328 · 2017: 286 · 2016: 256 · 2015: 187 · 2014: 161 · 2013: 28
- FWCI
- 94.43
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Science Foundation (U01 CA168370, P50 GM102706, 0540879)
- Howard Hughes Medical Institute
- National Institutes of Health (GM102706, GM081879)
- University of California, San Francisco (GM081879)
- National Cancer Institute (U01CA168370)
- National Institute of General Medical Sciences (P50GM081879, P50GM102706, GM081879, F32GM100611, GM102706)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR interference, dCas9, transcriptional repression, transcriptional activation, gene silencing, CRISPR/Cas9
- MeSH terms
- Bacterial Proteins, HeLa Cells, Humans, Saccharomyces cerevisiae, Streptococcus pyogenes, Gene Targeting, HEK293 Cells, RNA, Small Untranslated
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).
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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