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]
The cited source says what the claim attributes to it.
In the citing paper
“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).”
MethodInfluentialRNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429 · 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).”
BackgroundA monomeric red fluorescent protein
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., & Tsien, R. Y. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.082243699 · Cited by 2,346 · cited 2×
“We constructed a red fluorescent protein (mRFP)-based reporter system (Campbell et al., 2002) and inserted it into the E. coli MG1655 genome.”
BackgroundMethodEvolution and classification of the CRISPR–Cas systems
Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J. J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F. J. M., Wolf, Y. I., Yakunin, A. F., et al. (2011) · Nature Reviews Microbiology · doi:10.1038/nrmicro2577 · Cited by 2,331 · cited 1×
“About 40% of bacteria and 90%of archaea possessCRISPR/CRISPR-associated (Cas) systems to confer resistance to foreign DNA elements (Makarova et al., 2011).”
BackgroundEngineering and characterization of a superfolder green fluorescent protein
Pédelacq, J. D., Cabantous, S., Tran, T. H., Terwilliger, T. C., & Waldo, G. S. (2005) · Nature Biotechnology · doi:10.1038/nbt1172 · Cited by 2,295 · cited 2×
“The paused RNAP aborts transcription elongation upon encountering the dCas9-sgRNA roadblock. sfGFP (Pédelacq et al., 2006).”
BackgroundRNA-programmed genome editing in human cells
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013) · eLife · doi:10.7554/elife.00471 · Cited by 2,098 · 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).”
BackgroundTargeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2507 · Cited by 1,863 · 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).”
BackgroundRNA-guided genetic silencing systems in bacteria and archaea
Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012) · Nature · doi:10.1038/nature10886 · Cited by 1,851 · cited 1×
“CRISPR systems use small base-pairing RNAs to target and cleave foreign DNA elements in a sequence-specific manner (Wiedenheft et al., 2012).”
BackgroundSelf-citationLutz, R. (1997) · Nucleic Acids Research · doi:10.1093/nar/25.6.1203 · Cited by 1,694 · cited 1×
“The genes were PCR amplified and inserted into a vector containing an aTc-inducible promoter PLtetO-1 (Lutz and Bujard, 1997), a chloramphenicol-selectable marker, and a p15A replication origin.”
MethodProgramming cells by multiplex genome engineering and accelerated evolution
Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009) · Nature · doi:10.1038/nature08187 · Cited by 1,529 · cited 2×
“coli MG1655 strain was transformed with a temperature-sensitive plasmid pKD46 that contained l-Red recombination proteins (Exo, Beta, and Gam) (Wang et al., 2009).”
MethodCRISPR interference: RNA-directed adaptive immunity in bacteria and archaea
Marraffini, L. A., & Sontheimer, E. J. (2010) · Nature Reviews Genetics · doi:10.1038/nrg2749 · Cited by 919 · cited 1×
“See also Figure S1. determined by both sgRNA-DNA base pairing and a short DNA motif (protospacer adjacent motif [PAM] sequence: NGG) juxtaposed to the DNA complementary region (Marraffini and Sontheimer, 2010).”
BackgroundNascent transcript sequencing visualizes transcription at nucleotide resolution
Churchman, L. S., & Weissman, J. S. (2011) · Nature · doi:10.1038/nature09652 · Cited by 823 · cited 2×
“Nascent RNA was purified from the eluate using the miRNeasy kit (QIAGEN) and was converted to DNA using a previously established library generation protocol (Churchman and Weissman, 2011).”
MethodSelf-citationEfficient construction of sequence-specific TAL effectors for modulating mammalian transcription
Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., & Arlotta, P. (2011) · Nature Biotechnology · doi:10.1038/nbt.1775 · Cited by 712 · cited 1×
“RNA interference (RNAi) and engineered DNA-binding proteins such as zinc finger or transcription-activator-like effector (TALE) proteins, have emerged as powerful technologies for targeted gene regulation (Hannon, 2002; Beerli and Barbas, 2002; Zhang et al., 2011).”
BackgroundThe Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation
Klug, A. (2010) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-010909-095056 · Cited by 710 · cited 1×
“In addition, custom DNA-binding proteins, such as zinc finger or TALE proteins, remain somewhat difficult and expensive to design, develop, and empirically test in the cellular context (Klug, 2010).”
BackgroundEngineering polydactyl zinc-finger transcription factors
Beerli, R. R., & Barbas, C. F. (2002) · Nature Biotechnology · doi:10.1038/nbt0202-135 · Cited by 498 · cited 1×
“RNA interference (RNAi) and engineered DNA-binding proteins such as zinc finger or transcription-activator-like effector (TALE) proteins, have emerged as powerful technologies for targeted gene regulation (Hannon, 2002; Beerli and Barbas, 2002; Zhang et al., 2011).”
BackgroundStructures of the RNA-guided surveillance complex from a bacterial immune system
Wiedenheft, B., Lander, G. C., Zhou, K., Jore, M. M., Brouns, S. J. J., John van der, O. O. S. T., Doudna, J. A., & Nogales, E. (2011) · Nature · doi:10.1038/nature10402 · Cited by 404 · cited 1×
“Importantly, any single mutation of the first 7 nt dramatically decreased repression, suggesting that this sequence constitutes a ‘‘seed region’’ for binding, as noted previously for both the type I and type II CRISPR systems (Wiedenheft et al., 2011; Jinek et al., 2012; Gasiunas et al., 2012).”
BackgroundSelf-citationLucks, J. B., Mortimer, S., Trapnell, C., Luo, S. J., Aviran, S., Schroth, G. P., Pachter, L. S., Doudna, J. A., & Arkin, A. P. (2011) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1106501108 · Cited by 397 · cited 1×
“In the CRISPRi system, it may also be possible to link multiple sgRNAs into transcriptional circuits in which one upstream sgRNA controls the expression of a different downstream sgRNA (Lucks et al., 2011).”
Self-citationLewis, M. (2005) · Comptes Rendus Biologies · doi:10.1016/j.crvi.2005.04.004 · Cited by 260 · cited 1×
“We performed b-galactosidase assays to measure LacZ expression from the knockdown strains, with and without isopropyl b-D-1-thiogalactopyranoside (IPTG), a chemical that inhibits the lac repressor (LacI) (Lewis, 2005).”
MethodRNA processing enables predictable programming of gene expression
Qi, L. S., Haurwitz, R. E., Shao, W., Doudna, J. A., & Arkin, A. P. (2012) · Nature Biotechnology · doi:10.1038/nbt.2355 · Cited by 198 · cited 2×
“Genetic transformation and verification of transformationwere done as previously described (Qi et al., 2012), using AmpR, CmR, or KanR genes as selectable markers.”
BackgroundMethodSelf-citationDiscontinuous Mechanism of Transcription Elongation
Nudler, E., Goldfarb, A., & Kashlev, M. (1994) · Science · doi:10.1126/science.8047884 · Cited by 144 · cited 1×
“The distance between the pause site and the target site is 19 bp, which is in perfect accordance with the previously reported 18 bp distance between the nucleotide incorporation of RNAP and its front edge (Nudler et al., 1994).”
Background
Flagged first, then by citations
1 of the 30 references couldn’t be matched to a known paper.
Cited by
Sorted by citations
Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133
The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062
Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762
Gene regulation by long non-coding RNAs and its biological functions
Statello, L., Guo, C.-J., Chen, L.-L., & Huarte, M. (2021) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-020-00315-9 · Cited by 4,991
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
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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