The DOI matches its Crossref record; 46 of the 46 references it cites were found in a registry or an index; none of the 5 sources its claims cite is checked for support yet.
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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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
“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
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In 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: 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
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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
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“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.
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“…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
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Items 701–800 of 1,013
Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq
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“17,18 ) and a second-generation, dual-effector CRISPRi system, KRAB–dCas9– MeCP2 (refs.”
A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells
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“One application of the CRISPR/Cas9 system is based on a cat-alytically inactive form of Cas9 (dead Cas9, dCas9), which functions as a customized DNA-binding protein relying on gRNA sequences to regulate the transcription of a specific target sequence (13).”
BackgroundTruong, V. A., Hsu, M., Nguyen, N. T. K., Lin, M., Shen, C., Lin, C., & Hu, Y. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz267 · Cited by 78
“CRISPR activation (CRISPRa) was developed to stimulate target gene expression, by fusing dCas9 with transcription activators such as VP64 (9).”
BackgroundTranscriptional control by enhancers and enhancer RNAs
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“[177] Gilbert LA, Larson M, Morsut L, et al.”
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“This further suggests that the mechanism of repression may not involve steric hindrance of RNA polymerase II elongation, as postulated previously 38 , and instead may involve inhibition of transcription initiation.”
InfluentialPlacental imprinting: Emerging mechanisms and functions
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“CRISPRi screen Screens were performed in K562 cells stably expressing a constitutive SFFV-dCas9-BFP-KRAB CRISPRi transcriptional repression construct using lentiviral transduction [54] (Addgene plasmid # 46911).”
MethodSelf-citationVirus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing
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BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2
Samelson, A. J., Tran, Q. D., Robinot, R., Carrau, L., Rezelj, V. V., Kain, A. M., Chen, M., Ramadoss, G. N., Guo, X., Lim, S. A., et al. (2022) · Nature Cell Biology · doi:10.1038/s41556-021-00821-8 · Cited by 76
“We generated a polyclonal Calu-3 line constitutively expressing machinery to enable CRISPRi-based genetic screens 20,21 and validated its CRISPRi activity (Fig.”
Self-citationBossi, D., Cicalese, A., Dellino, G. I., Luzi, L., Riva, L., D’Alesio, C., Diaferia, G. R., Carugo, A., Cavallaro, E. C., Piccioni, R., et al. (2016) · Cancer Discovery · doi:10.1158/2159-8290.cd-15-1200 · Cited by 78
Genetic circuit characterization and debugging using RNA‐seq
Gorochowski, T. E., Borujeni, A. E., Park, Y., Nielsen, A. A. K., Zhang, J., Der, B. S., Gordon, D. B., & Voigt, C. A. (2017) · Molecular Systems Biology · doi:10.15252/msb.20167461 · Cited by 78
“Gates can be built based on many classes of biochemistry (e.g., DNA-binding proteins (Moon et al , 2012; Stanton et al , 2014), recombinases (Bonnet et al , 2013; Siuti et al , 2013; Fernandez-Rodriguez et al , 2015), and CRISPRi (Gilbert et al , 2013; Larson et al , 2013; Qi et al , 2013; Kiani et al , 2014; Nielsen…”
CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases
Koo, B., Kim, J., Kweon, J., Jin, C. E., Kim, S., Kim, Y., & Shin, Y. M. (2018) · Sensors and Actuators B Chemical · doi:10.1016/j.snb.2018.06.069 · Cited by 78
Next stop for the CRISPR revolution: RNA‐guided epigenetic regulators
Vora, S., Tuttle, M. L., Cheng, J. Y., & Church, G. M. (2016) · FEBS Journal · doi:10.1111/febs.13768 · Cited by 75
Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells
Pickar‐Oliver, A., Black, J. B., Lewis, M. M., Mutchnick, K. J., Klann, T. S., Gilcrest, K. A., Sitton, M. J., Nelson, C. E., Barrera, A., Bartelt, L. C., et al. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0235-7 · Cited by 77
CRISPR/Cas-Based Modifications for Therapeutic Applications: A Review
Bharathkumar, N., Abraham, S., Meera, P., Aksah, S., Muthu, K., Saravanan, K. M., & Anand, T. (2021) · Molecular Biotechnology · doi:10.1007/s12033-021-00422-8 · Cited by 80
Unraveling the 3D genome: genomics tools for multiscale exploration
Risca, V. I., & Greenleaf, W. J. (2015) · Trends in Genetics · doi:10.1016/j.tig.2015.03.010 · Cited by 77
The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex
Zeng, Y., Cui, Y., Zhang, Y., Zhang, Y., Liang, M., Chen, H., Lan, J., Song, G., & Lou, J. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx1117 · Cited by 79
In vivo locus-specific editing of the neuroepigenome
Yim, Y. Y., Teague, C. D., & Nestler, E. J. (2020) · Nature Reviews Neuroscience · doi:10.1038/s41583-020-0334-y · Cited by 76
Applications of CRISPR Genome Engineering in Cell Biology
Wang, F., & Qi, L. S. (2016) · Trends in Cell Biology · doi:10.1016/j.tcb.2016.08.004 · Cited by 79
“By fusing dCas9 to transcriptional repressors, such as the Kruppel-associated box (KRAB) domain, CRISPRi can efficiently repress coding and noncoding genes, such as miRNAs and large intergenic noncoding RNAs (lincRNAs) in mammalian cells [46,47,49,50].”
Self-citationDevelopment and application of CRISPR/Cas9 technologies in genomic editing
Zhang, C., Quan, R., & Wang, J. (2018) · Human Molecular Genetics · doi:10.1093/hmg/ddy120 · Cited by 80
Ng, S. R., Rideout, W. M., Akama‐Garren, E. H., Bhutkar, A. J., Mercer, K. L., Schenkel, J. M., Bronson, R. T., & Jacks, T. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1821893117 · Cited by 78
“The adaptation of CRISPR-Cas9 for transcriptional activation or inhibition (41 – 46) has further extended the utility of the CRISPR-Cas9 system for modeling additional gain-of-function mutations.”
Modulating gene regulation to treat genetic disorders
Matharu, N. K., & Ahituv, N. (2020) · Nature Reviews Drug Discovery · doi:10.1038/s41573-020-0083-7 · Cited by 79
CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics
Ghosh, D., Venkataramani, P., Nandi, S., & Bhattacharjee, S. (2019) · Cancer Cell International · doi:10.1186/s12935-019-0726-0 · Cited by 83
Evaluation of sgRNA Target Sites for CRISPR-Mediated Repression of TP53
Lawhorn, I. E. B., Ferreira, J. P., & Wang, C. L. (2014) · PLoS ONE · doi:10.1371/journal.pone.0113232 · Cited by 75
Breeding Major Cereal Grains through the Lens of Nutrition Sensitivity
Yu, S., & Tian, L. (2017) · Molecular Plant · doi:10.1016/j.molp.2017.08.006 · Cited by 77
Development and Application of CRISPR/Cas in Microbial Biotechnology
Ding, W., Zhang, Y., & Shi, S. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.00711 · Cited by 81
Strategies to target long non-coding RNAs in cancer treatment: progress and challenges
Dizaji, B. F. (2020) · Egyptian Journal of Medical Human Genetics · doi:10.1186/s43042-020-00074-4 · Cited by 78
O’Geen, H., Tomková, M., Combs, J. A., Tilley, E., & Segal, D. J. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac123 · Cited by 79
“We only observed this correlation in the expanded region (250 bp ± 1 kb), probably because H3K36me3 is associated with active transcription in gene bodies and is hence more prevalent downstream of TSS (4).”
BackgroundInfluentialCRISPR/Cas systems for the detection of nucleic acid and non-nucleic acid targets
Su, W., Li, J., Chen, J., Chen, C., Wang, Y., Dai, H., Li, F., & Liu, P. (2023) · Nano Research · doi:10.1007/s12274-023-5567-4 · Cited by 79
Synthetic Gene Circuits for Regulation of Next‐Generation Cell‐Based Therapeutics
Teixeira, A. P., & Fussenegger, M. (2023) · Advanced Science · doi:10.1002/advs.202309088 · Cited by 79
Genome Editing and Its Applications in Model Organisms
Ma, D., & Liu, F. (2015) · Genomics Proteomics & Bioinformatics · doi:10.1016/j.gpb.2015.12.001 · Cited by 81
“Soon after, dCas9 was fused to effector domains with distinct regulatory functions for stable and efficient repression or activation at the transcriptional level in human and yeast cells [68].”
BackgroundGolden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes
Vad‐Nielsen, J., Lin, L., Bolund, L., Nielsen, A. L., & Luo, Y. (2016) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-016-2271-5 · Cited by 72
“We co-transfected HEK293T cells with a plasmid expressing dSpCas9 coupled to a transcriptional repressor domain, KRAB, [6] and an equal molar amount of either the M10-CRISPRi containing five pairs of gRNAs, ten individual gRNA vectors, or an empty gRNA plasmid.”
BackgroundMethodFrom biomarkers to therapeutic targets—the promises and perils of long non-coding RNAs in cancer
Gutschner, T., Richtig, G., Haemmerle, M., & Pichler, M. (2017) · Cancer and Metastasis Reviews · doi:10.1007/s10555-017-9718-5 · Cited by 78
Can changes in histone acetylation contribute to memory formation?
López‐Atalaya, J. P., & Barco, Á. (2014) · Trends in Genetics · doi:10.1016/j.tig.2014.09.003 · Cited by 79
Diallo, I., & Provost, P. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21051627 · Cited by 74
“Thus, CRISPR interference (CRISPRi), similar to RNAi in concept but more robust in performance, uses a sgRNA and a version of the cas9 protein devoid of endonucleolytic activity (deadcas9 or dcas9) to mediate gene silencing [48].”
BackgroundEfficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus
Xu, K., Ren, C., Liu, Z., Zhang, T., Zhang, T., Li, D., Wang, L., Yan, Q., Guo, L., Shen, J., et al. (2014) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-014-1679-z · Cited by 78
“Actually, we have generated our hStCas9 nickases and dead Cas9 (dCas9) by introducing the mutations with D10A in RuvC or/and H847A in HNH [23, 24], all of the mutants abolished the DNA cleavage activity for hStCas9 in both yeast AH109 and human 293T cells (data not shown), suggesting additional potential applications…”
MethodSelf-citationMechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation
Mekler, V. M., Станиславович, М. Л., & Severinov, K. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1619926114 · Cited by 74
“A Cas9 derivative (dCas9) that lacks endonuclease activity but can still bind to targets (7) is used for transcriptome modulation (11) and visualization of genomic loci in live cells (12).”
BackgroundCRISPR-Cas9 genome engineering: Treating inherited retinal degeneration
Burnight, E. R., Giacalone, J. C., Cooke, J. A., Thompson, J. R., Bohrer, L. R., Chirco, K. R., Drack, A. V., Fingert, J. H., Worthington, K. S., Wiley, L. A., et al. (2018) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2018.03.003 · Cited by 79
dCas9-targeted locus-specific protein isolation method identifies histone gene regulators
Tsui, C., Inouye, C. J., Levy, M. J., Lu, A. C., Florens, L. A., Washburn, M. P., & Tjian, R. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1718844115 · Cited by 74
Perenthaler, E., Yousefi, S., Niggl, E., & Barakat, T. S. (2019) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2019.00352 · Cited by 75
“In this case, the enhancer adoption leads to adult-onset demyelinating leukodystrophy (ADLD), which is a progressive neurologic disorder affecting the myelination of the central nervous system (Giorgio et al., 2015).”
BackgroundApplications of CRISPR-Cas systems in lactic acid bacteria
Roberts, A., & Barrangou, R. (2020) · FEMS Microbiology Reviews · doi:10.1093/femsre/fuaa016 · Cited by 74
Gene Editing by Extracellular Vesicles
Kostyushev, D. S., Kostyusheva, A., Brezgin, S., Vv, S., Volchkova, E. V., Lukashev, A. N., & Chulanov, V. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21197362 · Cited by 75
“Alternatively, nuclease-null Cas9 (or dead Cas9; dCas) proteins may serve as carriers to bring enzymes or functionally active factors to certain locations in the genome [6,7].”
BackgroundInducible expression of large gRNA arrays for multiplexed CRISPRai applications
Shaw, W. M., Studená, L., Roy, K., Hapeta, P., McCarty, N. S., Graham, A. E., Ellis, T., & Ledesma‐Amaro, R. (2022) · Nature Communications · doi:10.1038/s41467-022-32603-7 · Cited by 73
Advances in Nanoparticles as Non-Viral Vectors for Efficient Delivery of CRISPR/Cas9
Kim, M., Hwang, Y., Lim, S., Jang, H., & Kim, H. (2024) · Pharmaceutics · doi:10.3390/pharmaceutics16091197 · Cited by 77
Synthetic lethal strategies for the development of cancer therapeutics
Ngoi, N. Y. L., Gallo, D., Torrado, C., Nardo, M., Durocher, D., & Yap, T. A. (2024) · Nature Reviews Clinical Oncology · doi:10.1038/s41571-024-00966-z · Cited by 81
“Examples of effector proteins include transcriptional activators (CRISPRa) 77–79 or repressors (CRISPRi) 80–82 , epigenetic regulators 83–90 , and systems capable of directly modifying DNA such as base editors 91,92 or prime editors 93–95 .”
CRISPR-Cas9 technology: applications and human disease modelling
Torres-Ruiz, R., & Rodríguez, S. (2016) · Briefings in Functional Genomics · doi:10.1093/bfgp/elw025 · Cited by 77
de la Fuente‐Núñez, C., & Lu, T. K. (2016) · Integrative Biology · doi:10.1039/c6ib00140h · Cited by 75
The virtuous cycle of human genetics and mouse models in drug discovery
Nadeau, J. H., & Auwerx, J. H. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-018-0009-9 · Cited by 78
Synthetic epigenetics—towards intelligent control of epigenetic states and cell identity
Jurkowski, T. P., Ravichandran, M., & Stepper, P. (2015) · Clinical Epigenetics · doi:10.1186/s13148-015-0044-x · Cited by 73
“Existing technologies used to modulate transcription of desired genes, like RNAi [31], gene knock-out experiments or expression of recombinant proteins (for example, overexpression of cDNA constructs), as well as programmable activators and silencers [32-36] require constitutive expression of the constructs to maintai…”
BackgroundExploiting CRISPR–Cas immune systems for genome editing in bacteria
Barrangou, R., & van Pijkeren, J. (2015) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2015.10.003 · Cited by 75
“In particular, variants of Cas9 in which both domains have been inactivated, generated a deactivated Cas9 (dCas9), constitute DNA binding proteins that can be used as transcriptional effectors when targeting promoter regions, either as repressors that block RNA polymerases, or fused to activators that enhance transcri…”
BackgroundLong Non-coding RNA DANCR as an Emerging Therapeutic Target in Human Cancers
Jin, S.-J., Jin, M., Xia, B.-R., & Jin, W. (2019) · Frontiers in Oncology · doi:10.3389/fonc.2019.01225 · Cited by 70
Functional Genomics for Cancer Drug Target Discovery
Haley, B., & Roudnicky, F. (2020) · Cancer Cell · doi:10.1016/j.ccell.2020.04.006 · Cited by 75
Cas13d: A New Molecular Scissor for Transcriptome Engineering
Gupta, R. K., Ghosh, A., Chakravarti, R., Singh, R., Ravichandiran, V., Swarnakar, S., & Ghosh, D. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.866800 · Cited by 74
“Utilization of Cas9 Beyond Genome Editing The type II CRISPR-Cas9 system is most widely used and most accepted because it is highly flexible to target and cleave any DNA sequence of the broad spectrum of species, to get an enviable editing outcome with unprecedented ease and specificity (Esvelt et al., 2013; Gilbert e…”
BackgroundA versatile, high-efficiency platform for CRISPR-based gene activation
Heidersbach, A. J., Dorighi, K. M., Gomez, J. A., Jacobi, A. M., & Haley, B. (2023) · Nature Communications · doi:10.1038/s41467-023-36452-w · Cited by 72
Modulating the expression of long non‐coding RNAs for functional studies
Liu, S. J., & Lim, D. A. (2018) · EMBO Reports · doi:10.15252/embr.201846955 · Cited by 74
“By localizing gene activation domains such as VP64 just upstream of the TSS of lncRNAs (or any other gene transcribed by RNA polymerase II) [91,93,95], lncRNA genes can be specifically overexpressed [94].”
A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans
Wensing, L. F., Sharma, J., Uthayakumar, D., Proteau, Y., Chavez, A., & Shapiro, R. S. (2019) · mSphere · doi:10.1128/msphere.00002-19 · Cited by 71
“be fused to dCas9 to significantly enhance target gene repression in human cells (52), and dCas9-Mxi1 fusions similarly enhance repression in Saccharomyces cerevisiae (52,”
BackgroundMethodInfluentialGenome engineering: a new approach to gene therapy for neuromuscular disorders
Nelson, C. E., Robinson-Hamm, J. N., & Gersbach, C. A. (2017) · Nature Reviews Neurology · doi:10.1038/nrneurol.2017.126 · Cited by 73
Tóth, E., Czene, B. C., Kulcsár, P. I., Krausz, S. L., Tálas, A., Nyeste, A., Varga, É., Huszár, K., Weinhardt, N., Ligeti, Z., et al. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky815 · Cited by 74
Hsu, M., Liao, H.-T., Truong, V. A., Huang, K.-L., Yu, F.-J., Chen, H., Nguyen, T. K. N., Макаревич, П. И., Parfyonova, Y. V., & Hu, Y.-C. (2019) · Theranostics · doi:10.7150/thno.36790 · Cited by 71
“The activation domain of VP64 gene was PCR-amplified from pHAGE EF1α dCas9-VP64 (Addgene #50918 [24]) and subcloned into pBac-dCas9, downstream of the dCas9 sequence, to generate pBac-dCas9_VP64.”
InfluentialCRISPR-based metabolic pathway engineering
Zhao, D., Zhu, X., Zhou, H., Sun, N., Wang, T., Bi, C., & Zhang, X. (2020) · Metabolic Engineering · doi:10.1016/j.ymben.2020.10.004 · Cited by 70
A functional genetic toolbox for human tissue-derived organoids
Sun, D., Evans, L., Perrone, F., Sokleva, V., Lim, K., Rezakhani, S., Lutolf, M., Zilbauer, M., & Rawlins, E. L. (2021) · eLife · doi:10.7554/elife.67886 · Cited by 71
“They have been rapidly adopted in cancer and pluripotent stem cell (PSC) lines (Bowden et al., 2020; Gilbert et al., 2013; Tian et al., 2019), but not in tissue-derived human organoids.”
BackgroundGold Nanomaterials‐Implemented CRISPR‐Cas Systems for Biosensing
Fu, R., & Xianyu, Y. (2023) · Small · doi:10.1002/smll.202300057 · Cited by 73
Optogenetic characterization methods overcome key challenges in synthetic and systems biology
Olson, E. J., & Tabor, J. J. (2014) · Nature Chemical Biology · doi:10.1038/nchembio.1559 · Cited by 71
Long, L., Guo, H., Yao, D., Xiong, K., Li, Y., Liu, P., Zhu, Z., & Liu, D. (2015) · Cell Research · doi:10.1038/cr.2015.35 · Cited by 71
CRISPR/Cas9-Based Genome Editing in Mice by Single Plasmid Injection
Fujihara, Y., & Ikawa, M. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00015-5 · Cited by 70
Pathak, G. P., Spiltoir, J. I., Höglund, C., Polstein, L. R., Heine-Koskinen, S., Gersbach, C., Rossi, J., & Tucker, C. L. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx260 · Cited by 68
Jing, X., Xie, B., Chen, L., Zhang, N., Jiang, Y., Qin, H., Wang, H., Hao, P., Yang, S., & Li, X. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky433 · Cited by 71
“Similar to the mutant Cas9 variant (dCas9) that is capable of binding target DNA but inactive in DNA cleavage (22,23), the LshCas13a R1278A mutant (referred to as dCas13a) bound target RNA more strongly (KD ∼ 7 nM) than the wild-type (KD ∼ 46 nM), although it was catalytically inactive (7).”
BackgroundBoulay, G., Volorio, A., Iyer, S., Broye, L. C., Stamenkovic, I., Riggi, N., & Rivera, M. N. (2018) · Genes & Development · doi:10.1101/gad.315192.118 · Cited by 75
“To resolve this issue, we designed an experimental strategy for testing the function of specific repeat enhancer sites by targeting immediately adjacent unique genomic sequenceswith CRISPR/dCas9-KRAB (Gilbert et al. 2013, 2014; Thakore et al. 2015; Klann et al. 2017).”
MethodEpigenome Engineering in Cancer: Fairytale or a Realistic Path to the Clinic?
Falahi, F., Sgro, A., & Blancafort, P. (2015) · Frontiers in Oncology · doi:10.3389/fonc.2015.00022 · Cited by 70
Basak, J., & Nithin, C. (2015) · Frontiers in Plant Science · doi:10.3389/fpls.2015.01001 · Cited by 72
“20 base pair guide RNAs (gRNA) are easy to design and can target the Cas9 protein to almost any desired region in the genome to bind to its DNA target by Watson-Crick base-pairing (Gilbert et al., 2013; Bortesi and Fischer, 2015).”
BackgroundThe HRI branch of the integrated stress response selectively triggers mitophagy
Chakrabarty, Y., Yang, Z., Chen, H., & Chan, D. C. (2024) · Molecular Cell · doi:10.1016/j.molcel.2024.01.016 · Cited by 74
Functional interpretation of non‐coding sequence variation: Concepts and challenges
Paul, D. S., Soranzo, N., & Beck, S. (2013) · BioEssays · doi:10.1002/bies.201300126 · Cited by 70
“For example, the CRISPR-associated catalytically inactive Cas9 protein, termed dCas9, can be fused to activator domains [69, 70], repressor domains [69] or potentially domains that alter different epigenetic states [71].”
BackgroundCas9-Based Tools for Targeted Genome Editing and Transcriptional Control
Xu, T., Li, Y., Nostrand, J. D. V., He, Z., & Zhou, J. Z. (2014) · Applied and Environmental Microbiology · doi:10.1128/aem.03786-13 · Cited by 71
The impact of CRISPR–Cas9 on target identification and validation
Moore, J. D. (2015) · Drug Discovery Today · doi:10.1016/j.drudis.2014.12.016 · Cited by 73
Nanotechnology based CRISPR/Cas9 system delivery for genome editing: Progress and prospect
Deng, H., Huang, W., & Zhang, Z. (2019) · Nano Research · doi:10.1007/s12274-019-2465-x · Cited by 70
“Modifying dCas9 with VP64-p65-Rta, SAM or Suntag activators to generate CRISPRa system could upregulate gene activation in mammalian cell types, while fusing Krüppel associated box (KRAB) to dCas9 was demonstrated to downregulate about 99% of target gene expression in human cells [24, 25].”
BackgroundMethodSynthetic Biological Circuits within an Orthogonal Central Dogma
Costello, A., & Badran, A. H. (2020) · Trends in biotechnology · doi:10.1016/j.tibtech.2020.05.013 · Cited by 71
“Programmable TFs have historically required the expression of orthogonal DNA-binding protein modules to regulate transcriptional circuits, incorporating zinc fingers (ZFs) (Figure 3C) [58] or transcription activator-like effectors (TALEs) (Figure 3D) [59] fused to relevant activating [60–63] or repressive [64] effecto…”
BackgroundCRISPR–Cas system: a powerful tool for genome engineering
Liu, L., & Fan, X.-D. (2014) · Plant Molecular Biology · doi:10.1007/s11103-014-0188-7 · Cited by 73
“In addition to genome editing, CRISPR interference (CRISPRi) can efficiently and selectively repress or activate transcription of targeted genes using a modified Cas9 protein lacking endonucleolytic activity (Qi et al. 2013; Gilbert et al. 2013; Larson et al. 2013) (Fig.”
BackgroundRNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?
Gasiūnas, G., & Šikšnys, V. (2013) · Trends in Microbiology · doi:10.1016/j.tim.2013.09.001 · Cited by 70
Dong, F., Xie, K., Chen, Y., Yang, Y., & Mao, Y. (2016) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2016.11.129 · Cited by 75
Gene repression via multiplex gRNA strategy in Y. lipolytica
Zhang, J., Peng, Y.-Z., Liu, D., Liu, H., Cao, Y., Li, B., Li, C., & Yuan, Y. (2018) · Microbial Cell Factories · doi:10.1186/s12934-018-0909-8 · Cited by 71
“The engineered CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system has been proved its ability of highly selective transcriptional modulation over a significant dynamic range [36, 37].”
BackgroundMethodWhat has single-cell RNA-seq taught us about mammalian spermatogenesis?
Suzuki, S., Diaz, V. D., & Hermann, B. P. (2019) · Biology of Reproduction · doi:10.1093/biolre/ioz088 · Cited by 70
“Likewise, leveraging CRISPR/Cas9 to delete key novel target [136, 137], along with CRISPR activation [138–142], CRISPR inhibition [143, 144], and CRISPR epigenetic modification [145–153], which will define the molecular circuitry controlling cell fate in the spermatogenic lineage.”
BackgroundGrimmer, M., Stolzenburg, S., Ford, E., Lister, R., Blancafort, P., & Farnham, P. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku708 · Cited by 69
High-Throughput Approaches to Pinpoint Function within the Noncoding Genome
Montalbano, A., Canver, M. C., & Sanjana, N. E. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.09.017 · Cited by 68
“In mammalian systems, dCas9 itself is not sufficient for repression, and effective repression has required a fusion of dCas9 with the Kr€uppel-associated box (KRAB) effector domain (Gilbert et al., 2013).”
Wong, N. K., Huang, C., Islam, R., & Yip, S. P. (2018) · Journal of Hematology & Oncology · doi:10.1186/s13045-018-0673-6 · Cited by 70
Robust and stable transcriptional repression in Giardia using CRISPRi
McInally, S. G., Hagen, K. D., Nosala, C., Williams, J. C., Nguyen, K., Booker, J., Jones, K., & Dawson, S. C. (2018) · Molecular Biology of the Cell · doi:10.1091/mbc.e18-09-0605 · Cited by 73
“In both bacteria and human cell lines, targeting gRNAs close to the translation start site results in stronger repression (Gilbert et al., 2013; Qi et al., 2013).”
BackgroundMethodGao, Q., Ouyang, W., Kang, B., Han, X., Xiong, Y., Ding, R., Li, Y., Wang, F., Huang, L., Chen, L., et al. (2020) · Theranostics · doi:10.7150/thno.42325 · Cited by 72
“The AAVS1 locus in the first intron of the 144 PPP1R12C gene is one of the most commonly used genomic safe harbor (GSH) sites in human cell research [28], and 145 TITIN encoded by TTN gene is one of the myofibrillar proteins thought to play an important role in the assembly and 146 function of muscle sarcomeres [29].”
BackgroundEditing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation
Rao, S., Yao, Y., & Bauer, D. E. (2021) · Genome Medicine · doi:10.1186/s13073-021-00857-3 · Cited by 72
“Fusing a strong repressor, such as Kruppel-associated Box (KRAB) [69] and DNMT3A/3 L [70], to dCas9, results in stronger gene repression than dCas9 alone.”
BackgroundInfluentialPregnancy, preeclampsia and maternal aging: From epidemiology to functional genomics
Miller, E. C., Wilczek, A., Bello, N. A., Tom, S. E., Wapner, R. J., & Suh, Y. (2021) · Ageing Research Reviews · doi:10.1016/j.arr.2021.101535 · Cited by 78
Gene editing and its applications in biomedicine
Li, G., Li, X., Zhuang, S., Wang, L., Zhu, Y., Chen, Y., Sun, W. C., Wu, Z., Zhou, Z. L., Chen, J., et al. (2022) · Science China Life Sciences · doi:10.1007/s11427-021-2057-0 · Cited by 66
“transcriptional modulators (Gilbert et al., 2013; Qi et al., 2013) or epigenome-modifying factors (Liu et al.”
BackgroundCRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products
Fu, R., Wang, Y., Liu, Y., Liu, H., Zhao, Q., Zhang, Y., Wang, C., Li, Z., Jiao, B., & He, Y. (2022) · Food Chemistry · doi:10.1016/j.foodchem.2022.132919 · Cited by 68
CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy
Yao, S., He, Z., & Chen, C. (2015) · Human Gene Therapy · doi:10.1089/hum.2015.067 · Cited by 70
“89,90 94 Histone methyltransferase domains from G9A and Suv39H1 have been fused to zinc finger proteins, which led to local methylation of histone H3K9 and the consequent repression of target gene expression.”
CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii
Sidik, S. M., Huet, D., & Lourido, S. (2018) · Nature Protocols · doi:10.1038/nprot.2017.131 · Cited by 68
“However, CRISPR activation and CRISPR inhibition, two techniques that use cata-lytically inactive Cas9 to modulate transcription without causing DNA breaks, may overcome this hurdle 33,34 .”
Drug Inducible CRISPR/Cas Systems
Zhang, J., Chen, L., Zhang, J., & Wang, Y. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.07.015 · Cited by 77
Deaner, M., Mejia, J., & Alper, H. S. (2017) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00163 · Cited by 67
Gene-Specific Targeting of DNA Methylation in the Mammalian Genome
Urbano, A., Smith, J., Weeks, R. J., & Chatterjee, A. (2019) · Cancers · doi:10.3390/cancers11101515 · Cited by 67
“However, for robust epigenetic modifications, stable transduction and long-term expression of the dCas9 complex is likely to be necessary, especially when applying these systems to primary cells or pluripotent cells [93,116,117].”
BackgroundYeast metabolic engineering for the production of pharmaceutically important secondary metabolites
Rahmat, E., & Kang, Y. (2020) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-020-10587-y · Cited by 67
“In addition to CRISPR-Cas9, other CRISPR-related tools, such as CRISPR 237 interference and CRISPRmediated activation using dCas9, have been developed to modulate gene activation, gene expression, and expression of complex gene (Gilbert et al. 2013).”
MethodGenome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies
McTague, A., Rossignoli, G., Ferrini, A., Barral, S., & Kurian, M. A. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.630600 · Cited by 67
“This CRISPR-based transcriptional modulation is achieved by repressor or activator transcription domains fused to a catalytically inactive Cas9 (dCas9) and guide RNAs directed to the promoter or regulatory regions of specific genes (Gilbert et al., 2013).”
BackgroundCRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells
Bloomer, H., Khirallah, J., Li, Y., & Xu, Q. (2021) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2021.114087 · Cited by 75
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
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