The DOI matches its Crossref record; 29 of the 30 references it cites were found in a registry or an index.
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.
In the citing paper
“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]
The cited source says what the claim attributes to it.
In the citing paper
“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
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 · cited 6×
“Cell 152, 1173–1183, February 28, 2013 ª2013 Elsevier Inc. 1175 (Jinek et al., 2012), alleviated this lethality, as verified by transformation efficiency and E. coli growth rates (Figures S2A and S2B).”
BackgroundInfluentialSelf-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×
“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).”
BackgroundMapping and quantifying mammalian transcriptomes by RNA-Seq
Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., & Wold, B. (2008) · Nature Methods · doi:10.1038/nmeth.1226 · Cited by 13,696 · cited 1×
“To evaluate the specificity of CRISPRi on a genome-wide scale, we performed whole-transcriptome shotgun sequencing (RNAseq) of dCas9-transformed cells with and without sgRNA coexpression (Figure 4A) (Mortazavi et al., 2008).”
MethodRNA-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 3×
“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×
“The CRISPR (clustered regularly interspaced short palindromic repeats) system provides a potential platform for targeted gene regulation (Barrangou et al., 2007).”
BackgroundHannon, G. J. (2002) · Nature · doi:10.1038/418244a · Cited by 3,651
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
Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1208507109 · Cited by 2,769 · cited 2×
“…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).”
BackgroundZamore, P. D., Tuschl, T., Sharp, P. A., & Bartel, D. P. (2000) · Cell · doi:10.1016/s0092-8674(00)80620-0 · Cited by 2,751 · cited 1×
“This mechanism is distinct from that of RNAi, for which knockdown of gene expression requires the destruction of already transcribed messenger RNAs prior to their translation (Zamore et al., 2000).”
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
Flagged first, then by citations
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Items 1,001–1,026 of 1,026
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology
Gale, G. A. R., Schiavon Osorio, A. A., Mills, L. A., Wang, B., Lea-Smith, D. J., & McCormick, A. J. (2019) · Microorganisms · doi:10.3390/microorganisms7100409 · Cited by 61
Characterization and Repurposing of Type I and Type II CRISPR–Cas Systems in Bacteria
Hidalgo-Cantabrana, C., Goh, Y. J., & Barrangou, R. (2019) · Journal of Molecular Biology · doi:10.1016/j.jmb.2018.09.013 · Cited by 63
Combining CRISPRi and metabolomics for functional annotation of compound libraries
Anglada-Girotto, M., Handschin, G., Ortmayr, K., Campos, A. I., Gillet, L., Manfredi, P., Mulholland, C. V., Berney, M., Jenal, U., Picotti, P., et al. (2022) · Nature Chemical Biology · doi:10.1038/s41589-022-00970-3 · Cited by 60
Recent Progress and Challenges in CRISPR-Cas9 Engineered Algae and Cyanobacteria
Patel, V. K., Das, A., Kumari, R., & Kajla, S. (2023) · Algal Research · doi:10.1016/j.algal.2023.103068 · Cited by 50
Gao, Z., Harwig, A., Berkhout, B., & Herrera-Carrillo, E. (2017) · Transcription · doi:10.1080/21541264.2017.1322170 · Cited by 47
Amann, T., Schmieder, V., Faustrup Kildegaard, H., Borth, N., & Andersen, M. R. (2019) · Biotechnology and Bioengineering · doi:10.1002/bit.27101 · Cited by 46
“Additionally, reversible gene activation as well as repression can be achieved by using a catalytically dead Cas9 (dCas9) fused to an effector domain (Qi et al., 2013).”
G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation
Deng, H., Xu, H., Wang, Y., Jia, R., Ma, X., Feng, Y., & Chen, H. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad178 · Cited by 45
“The nuclease-deficient variants of dCas9 fused with a transcriptional activator (VP64) have been a versatile tool for controlling gene expression (54,55).”
Pattharaprachayakul, N., Lee, M., Incharoensakdi, A., & Woo, H. M. (2020) · Enzyme and Microbial Technology · doi:10.1016/j.enzmictec.2020.109619 · Cited by 43
Genome engineering of stem cell organoids for disease modeling
Sun, Y., & Ding, Q. (2017) · Protein & Cell · doi:10.1007/s13238-016-0368-0 · Cited by 39
“…regulate gene expression at the transcriptional levels (Cheng et al., 2013; Gilbert et al., 2013; Perez-Pinera et al., 2013a; Pinera et al., 2013b; Qi et al., 2013); with epigenetic regulators (e.g. acetyltransferase and histone demethylase) to manipulate the epi-status of a genomic region…”
BackgroundShen, Q., Ruan, H., Zhang, H., Wu, T., Zhu, K., Han, W., Dong, R., Ming, T., Qi, H., & Zhang, Y. (2024) · Frontiers in Microbiology · doi:10.3389/fmicb.2024.1375120 · Cited by 38
Lo, Y.-H., Horn, H. T., Huang, M.-F., Yu, W.-C., Young, C.-M., Liu, Q., Tomaske, M., Towers, M., Dominguez, A., Bassik, M. C., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-62818-3 · Cited by 37
“For example, CRISPRi (interference) 23 – 25 and CRISPRa (activation) 26 – 30 technologiesutilizecatalyticallyinactiveCas9(dCas9)fusedtoeithera transcriptional repressor (e.g., KRAB, Krüppel associated box) or a transcriptional activator (e.g., VPR, VP64-p65-Rta).”
Self-citationLohia, A., Sahel, D. K., Salman, M., Singh, V., Mariappan, I., Mittal, A., & Chitkara, D. (2022) · Asian Journal of Pharmaceutical Sciences · doi:10.1016/j.ajps.2022.02.001 · Cited by 33
“[17] Qi LS , Larson MH , Gilbert LA , Doudna JA , Weissman JS , Arkin AP , et al.”
BackgroundHeterologous production of cyanobacterial compounds
Dhakal, D., Chen, M., Luesch, H., & Ding, Y. (2021) · Journal of Industrial Microbiology and Biotechnology · doi:10.1093/jimb/kuab003 · Cited by 33
Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages
Piya, D., Nolan, N., Moore, M. L., Ramirez Hernandez, L. A., Cress, B. F., Young, R., Arkin, A. P., & Mutalik, V. K. (2023) · PLOS Biology · doi:10.1371/journal.pbio.3002416 · Cited by 31
“A catalytically inactive or “dead” Cas protein (such as dCas9 or dCas12a) enables programmable transcriptional knockdown (by binding to DNA and forming a transcriptional road block) yielding a loss-of-function phenotype in a DNA sequence–dependent manner [36–41].”
BackgroundSelf-citationDirect-seq: programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen
Song, Q., Ni, K., Liu, M., Li, Y., Wang, L., Wang, Y., Liu, Y., Yu, Z., Qi, Y., Lu, Z., et al. (2020) · Genome Biology · doi:10.1186/s13059-020-02044-w · Cited by 26
Hamilton, P. J., Lim, C. J., Nestler, E. J., & Heller, E. A. (2018) · Methods in Molecular Biology · doi:10.1007/978-1-4939-7774-1_5 · Cited by 28
A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept
Kirtania, P., Hódi, B., Mallick, I., Vass, I. Z., Fehér, T., Vass, I., & Kós, P. B. (2019) · PLOS ONE · doi:10.1371/journal.pone.0225375 · Cited by 22
“coli [2, 3, 5] and being used for wide variety of purposes in genetic engineering and synthetic biology [6].”
BackgroundMethodInfluentialWang, Y., Tian, Y., Xu, D., Cheng, S., Li, W.-W., & Song, H. (2025) · Biotechnology Advances · doi:10.1016/j.biotechadv.2025.108516 · Cited by 17
Hybrid multitask learning reveals sequence features driving specificity in the CRISPR/Cas9 system
Vora, D. S., Yadav, S., & Sundar, D. (2023) · Biomolecules · doi:10.3390/biom13040641 · Cited by 17
The epigenome editors: How tools such as CRISPR offer new details about epigenetics
Willyard, C. (2017) · Nature Medicine · doi:10.1038/nm0817-900 · Cited by 13
CRISPR-Cas mediated genome engineering of cyanobacteria
Sengupta, A., Liu, D., & Pakrasi, H. B. (2022) · Methods in Enzymology · doi:10.1016/bs.mie.2022.07.023 · Cited by 13
Direct capture of CRISPR guides enables scalable, multiplexed, and multi-omic Perturb-seq
Replogle, J. M., Xu, A., Norman, T. M., Meer, E. J., Terry, J. M., Riordan, D., Srinivas, N., Mikkelsen, T. S., Weissman, J. S., & Adamson, B. (2018) · doi:10.1101/503367 · Cited by 465
Self-citationXie, H., Bourgade, B., Stensjö, K., & Lindblad, P. (2025) · Microbial Cell Factories · doi:10.1186/s12934-025-02727-8 · Cited by 11
“Derived from the CRISPR-Cas system, the CRISPR interference-deactivated Cas (CRISPRi-dCas) system, lacking cleavage activity while retaining the ability to bind to target DNA using a single guide RNA, stands out as a preferred tool for fine-tuning gene expression [36].”
The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories
Opel, F., Axmann, I. M., & Klähn, S. (2022) · Advances in Biochemical Engineering/Biotechnology · doi:10.1007/10_2022_210 · Cited by 7
CRISPRi-assisted metabolic engineering of cyanobacteria for photosynthetic hyaluronic acid from CO2
Son, J., Lee, H. J., & Woo, H. M. (2025) · Journal of Biological Engineering · doi:10.1186/s13036-025-00494-z · Cited by 5
Cyanobacterial type I CRISPR-Cas systems: distribution, mechanisms, and genome editing applications
Zhang, Y., Yang, S., Zheng, X., & Tan, X. (2025) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2025.1552030 · Cited by 3
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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