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Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Open accessAbstract
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
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“CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) employ catalytically deactivated forms of Cas9 (dCas9) that are fused to transcriptional repressors or activators 29,101 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [29]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“The use of CRISPR/Cas9 system enables regulation of gene expression and make epigenetic alterations without introducing DNA double-strand breaks.428”
From Epigenetic regulation in major depression and other stress-related disorders: molecular mechanisms, clinical relevance and therapeutic potential (2023) · cites this paper as [428]
Experimental — accuracy not yet measuredThe 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 [25]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
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“Alternatives to DNA cleavage-induced editing include using CRISPR-Cas9 for direct chemical sequence alteration (base editing) 25,26 , providing RNA templates for gene alteration (prime editing) 27,28 , and for transcriptional control (CRISPR interference, CRISPRi; CRISPR activation, CRISPRa) 29,30 (Fig. 3).”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [29]
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“Due to its catalytically inactive nature, modifications of gene expression caused by dCas9 are transient, since the genomic DNA is not altered [61,77].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [77]
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“When both RuvC and HNH catalytic domains of Cas9 are modified through two silencing mutations, the system loses its DNA cleaving capabilities but retains the ability to bind to targeted sequences [61,77].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [77]
In the citing paper
“dCas9 can be repurposed as a site-specific DNA-binding domain transporting various functional domains to the target locus”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [17]
In the citing paper
“Moreover, the mutant virus that is formed in P1 cannot be passed to the P2 virus in equal proportions, which illustrates the steric hindrance caused by the association of the Cas9 protein with specific DNA sequences [34], and the time that elapses during cleavage and repair may affect viral genome packaging.”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [34]
In 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 [14]
In the citing paper
“Furthermore, a Cas9 mutant totally defective in endonuclease activity together with a guide RNA can specifically interfere with gene transcription without inducing any gene mutation in a process called CRISPR interference (CRISPRi) [89].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [89]
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Citations
Cites
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“While our paper was under review, several very recent studies reported the use of wild-type Cas9/CRISPR for targeted genome editing in diverse organisms (Mali et al., 2013; Cong et al., 2013; Jinek et al., 2013; Jiang et al., 2013; Hwang et al., 2013; Cho et al., 2013).”
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MethodRNA-Guided Human Genome Engineering via Cas9
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“While our paper was under review, several very recent studies reported the use of wild-type Cas9/CRISPR for targeted genome editing in diverse organisms (Mali et al., 2013; Cong et al., 2013; Jinek et al., 2013; Jiang et al., 2013; Hwang et al., 2013; Cho et al., 2013).”
BackgroundCRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., & Horvath, P. (2007) · Science · doi:10.1126/science.1138140 · Cited by 5,947 · cited 1×
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Efficient genome editing in zebrafish using a CRISPR-Cas system
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“…pyogenes: only a single gene encoding the Cas9 protein and two RNAs, a mature CRISPR RNA (crRNA) and a partially complementary trans-acting RNA (tracrRNA), are necessary and sufficient for RNA-guided silencing of foreign DNAs (Figure S1 available online) (Jinek et al., 2012; Gasiunas et al., 2012).”
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CRISPR RNA Maturation by Trans-Encoded Small RNA and Host Factor RNase III
Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., Eckert, M. R., Vogel, J., & Charpentier, E. (2011) · Nature · doi:10.1038/nature09886 · Cited by 2,563 · cited 3×
“Maturation of crRNA requires tracrRNA and RNase III (Deltcheva et al., 2011).”
MethodInfluential
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
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Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
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Gene regulation by long non-coding RNAs and its biological functions
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CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
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“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).”
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CRISPR-Cas systems for editing, regulating and targeting genomes
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Self-citationCrystal structure of Cas9 in complex with guide RNA and target DNA
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CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing
Labun, K., Montague, T. G., Krause, M., Cleuren, Y. N. T., Tjeldnes, H., & Valen, E. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz365 · Cited by 2,075
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
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“…to implement with applications involving multiplex 12–14 or genome-wide libraries of gRNAs 15, 16 and, unlike the paired nickase strategy, can also be used to improve the specificities of dCas9 25 or dCas9 fusions to heterologous effector domains such as transcriptional regulatory domains 17–19 .”
MethodDixit, A., Parnas, O., Li, B., Chen, J., Fulco, C. P., Jerby-Arnon, L., Marjanovic, N. D., Dionne, D., Burks, T., Raychowdhury, R., et al. (2016) · Cell · doi:10.1016/j.cell.2016.11.038 · Cited by 1,863
Self-citationDynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System
Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J. S., Blackburn, E. H., Weissman, J. S., Qi, L. S., et al. (2013) · Cell · doi:10.1016/j.cell.2013.12.001 · Cited by 1,988
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DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
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The CRISPR tool kit for genome editing and beyond
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Highly efficient Cas9-mediated transcriptional programming
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RNA virus interference via CRISPR/Cas13a system in plants
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“However, like dCas9, dCas13a retains its ability to bind specifically to the target RNA and therefore can function as an RNA-guided RNA-binding protein [11, 14].”
BackgroundLi, J., Norville, J. E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G. M., & Sheen, J. (2013) · Nature Biotechnology · doi:10.1038/nbt.2654 · Cited by 1,533
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“A number of clever strategies that could eventually be applied clinically involve the use of RNA-guided catalytically inactive Cas9 (“dead Cas9” or dCas9) to turn genes on and off by blocking transcriptional machinery or recruiting epigenetic regulators (59, 60).”
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RNA-guided gene activation by CRISPR-Cas9-based transcription factors
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Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system
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“coli (24, 35), which separated cas9 and the sgRNA in pCas and pTarget series, respectively (Fig.”
BackgroundMethodGTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling
Kraft, V. A. N., Bezjian, C. T., Pfeiffer, S., Ringelstetter, L., Müller, C., Zandkarimi, F., Merl‐Pham, J., Bao, X., Anastasov, N., Kössl, J., et al. (2019) · ACS Central Science · doi:10.1021/acscentsci.9b01063 · Cited by 1,367
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Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
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Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
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Editing DNA Methylation in the Mammalian Genome
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“…activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013).”
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High-throughput functional genomics using CRISPR-Cas9
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Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
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Self-citationThe chromatin accessibility landscape of primary human cancers
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“To verify a regulatory interaction for the predicted peak-to-gene links, we used a CRISPR interference (CRISPRi) (40) strategy using a catalytically dead Cas9 (dCas9) fused to a Kruppelassociated box (KRAB) domain, which mediates focal heterochromatin formation and functional silencing of noncoding DNA regulatory elem…”
MethodCRISPR interference (CRISPRi) for sequence-specific control of gene expression
Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., & Qi, L. S. (2013) · Nature Protocols · doi:10.1038/nprot.2013.132 · Cited by 1,241
Self-citationAdamson, B., Norman, T. M., Jost, M., Cho, M. Y., Nuñez, J. K., Chen, Y., Villalta, J. E., Gilbert, L. A., Horlbeck, M. A., Hein, M. Y., et al. (2016) · Cell · doi:10.1016/j.cell.2016.11.048 · Cited by 1,193
“coli MG1655-derived strain by lambda Red recombinase-mediated recombineering as described previously (Qi et al., 2013), and moved from the MG1655-derived strain into the dCas9expressing BW25113 strain by P1 transduction and selection on kanamycin following established protocols.”
MethodInfluentialSelf-citationCas9 as a versatile tool for engineering biology
Mali, P., Esvelt, K. M., & Church, G. M. (2013) · Nature Methods · doi:10.1038/nmeth.2649 · Cited by 1,197
The CRISPR/Cas9 system for plant genome editing and beyond
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Delivering CRISPR: a review of the challenges and approaches
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“Qu et al. repurposed CRISPR into an RNA-guided platform for controlling gene expression by developing a catalytically-dead Cas9 enzyme (dCas9) that retained its capability to recognize and bind a target DNA sequence (Qi et al., 2013).”
BackgroundCRISPR RNA–guided activation of endogenous human genes
Maeder, M. L., Linder, S. J., Cascio, V. M., Fu, Y., Ho, Q. H., & Joung, J. K. (2013) · Nature Methods · doi:10.1038/nmeth.2598 · Cited by 1,249
The promise and challenge of therapeutic genome editing
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Self-citationCRISPR/Cas9 in Genome Editing and Beyond
Wang, H., Russa, M. F. L., & Qi, L. S. (2016) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-060815-014607 · Cited by 1,213
“Although CRISPRi is generally highly efficient in prokaryotes, the dCas9–sgRNA complex alone may not be very efficient at silencing gene expression in mammalian cells (41).”
BackgroundInfluentialSelf-citationBikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. A. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt520 · Cited by 1,145
Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities
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CRISPR technology: A decade of genome editing is only the beginning
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“As a result, the first examples of engineered CRISPR-Cas involved transcriptional repression or activation to silence or up-regulate specific genes (8, 9).”
BackgroundSelf-citationGenetic circuit design automation
Nielsen, A. A. K., Der, B. S., Shin, J., Vaidyanathan, P., Paralanov, V., Strychalski, E. A., Ross, D., Densmore, D. M., & Voigt, C. A. (2016) · Science · doi:10.1126/science.aac7341 · Cited by 1,012
“Within this paradigm, the regulators performing the gate biochemistry could be transcrip-tionfactors( 21 , 63 ),RNA-basedregulation( 64 – 66 ), protein-protein interactions ( 19 , 67 ), CRISPR/ Cas-based regulation ( 68 – 71 ), or recombinases ( 25 , 26 , 72 ).”
Horlbeck, M. A., Gilbert, L. A., Villalta, J. E., Adamson, B., Pak, R. A., Chen, Y., Fields, A. P., Park, C. Y., Corn, J. E., Kampmann, M., et al. (2016) · eLife · doi:10.7554/elife.19760 · Cited by 894
“…toxicity due to nuclease activity, combined with our previous findings that CRISPRi enables inducible, reversible, and homogenous manipulation of gene expression (Gilbert et al., 2014; Mandegar et al., 2016; Qi et al., 2013), make CRISPRi a state-of-the-art approach for loss-of-function studies.”
BackgroundMethodInfluentialSelf-citationExploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials
Bikard, D., Euler, C. W., Jiang, W., Nussenzweig, P. M., Goldberg, G. W., Duportet, X., Fischetti, V. A., & Marraffini, L. A. (2014) · Nature Biotechnology · doi:10.1038/nbt.3043 · Cited by 986
CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes
Komor, A. C., Badran, A. H., & Liu, D. R. (2016) · Cell · doi:10.1016/j.cell.2016.10.044 · Cited by 1,037
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
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Applications of CRISPR technologies in research and beyond
Barrangou, R., & Doudna, J. A. (2016) · Nature Biotechnology · doi:10.1038/nbt.3659 · Cited by 1,005
Self-citationCHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering
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Metadata
- Authors
- Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A.
- Affiliations
- QB3 (US)
- Corresponding author
- Qi, Lei S.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2013-02
- Volume
- 152
- Issue
- 5
- Pages
- 1173-1183
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC3664290
- OpenAlex
- W2160051211
- Cited by
- 5,016
- References
- 30
- Citations by year
- 2026: 291 · 2025: 375 · 2024: 418 · 2023: 403 · 2022: 497 · 2021: 465 · 2020: 499 · 2019: 444 · 2018: 417 · 2017: 382 · 2016: 315 · 2015: 229 · 2014: 189 · 2013: 87
- FWCI
- 121.73
- Type
- journal article
- Language
- en
- Update notices
- erratum (2021-02-04)
- 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
- Howard Hughes Medical Institute
- National Cancer Institute (U01CA168370)
- National Institute of General Medical Sciences (P50GM081879, F32GM100611)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR interference, Cas9, transcriptional repression, dCas9, guide RNA, RNA polymerase binding, transcription factor binding, Escherichia coli, mammalian cells, off-target effects
- MeSH terms
- Endodeoxyribonucleases, Escherichia coli, Streptococcus pyogenes, Gene Expression, RNA Interference, Gene Knockdown Techniques, RNA, Small Untranslated, Transcription Initiation, Genetic, Transcription Elongation, Genetic
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