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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.
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]
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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
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BackgroundInfluentialSelf-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
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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).”
BackgroundMapping and quantifying mammalian transcriptomes by RNA-Seq
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“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×
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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
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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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“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
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Efficient genome engineering approaches for the short-lived African turquoise killifish
Harel, I., Valenzano, D. R., & Brunet, A. (2016) · Nature Protocols · doi:10.1038/nprot.2016.103 · Cited by 89
Beyond Native Cas9: Manipulating Genomic Information and Function
Mitsunobu, H., Teramoto, J., Nishida, K., & Kondo, A. (2017) · Trends in biotechnology · doi:10.1016/j.tibtech.2017.06.004 · Cited by 87
CRISPR-Cas based antiviral strategies against HIV-1
Wang, G., Zhao, N., Berkhout, B., & Das, A. T. (2017) · Virus Research · doi:10.1016/j.virusres.2017.07.020 · Cited by 86
Bioengineering Solutions for Manufacturing Challenges in CAR T Cells
Piscopo, N. J., Mueller, K. P., Das, A., Hematti, P., Murphy, W. L., Palecek, S. P., Capitini, C. M., & Saha, K. (2017) · Biotechnology Journal · doi:10.1002/biot.201700095 · Cited by 93
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“In particular, the advent of clustered regularly interspersed short palindromic repeat interference (CRISPRi), which requires only the dCas9 enzyme, which is defunct in endo-nucleolytic activity because of point mutations in the RuvC and HNH domains, and a custom single guide RNA (sgRNA) that conveys the DNA binding s…”
BackgroundJoly, J. H., Delfarah, A., Phung, P. S., Parrish, S., & Graham, N. A. (2019) · Journal of Biological Chemistry · doi:10.1074/jbc.ra119.011471 · Cited by 88
“Knockdown and overexpression of genes using CRISPR interference (38) and CRISPR activation (29) were done as described previously.”
BackgroundMethodInfluentialInnovations in 3D Tissue Models of Human Brain Physiology and Diseases
Lovett, M. L., Nieland, T. J. F., Dingle, Y. L., & Kaplan, D. L. (2020) · Advanced Functional Materials · doi:10.1002/adfm.201909146 · Cited by 87
Sapozhnikov, D. M., & Szyf, M. (2021) · Nature Communications · doi:10.1038/s41467-021-25991-9 · Cited by 83
CRISPR/Cas13a‐Based MicroRNA Detection in Tumor‐Derived Extracellular Vesicles
Hong, J., Son, T., Castro, C. M., & Im, H. (2023) · Advanced Science · doi:10.1002/advs.202301766 · Cited by 89
Cress, B. F., Jones, J. A., Kim, D. C., Leitz, Q. D., Englaender, J. A., Collins, S. M., Linhardt, R. J., & Koffas, M. A. G. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw231 · Cited by 93
Kim, J. (2016) · Nature Protocols · doi:10.1038/nprot.2016.104 · Cited by 89
Westbrook, A. W., Ren, X., Moo‐Young, M., & Chou, C.-H. P. (2017) · Biotechnology and Bioengineering · doi:10.1002/bit.26459 · Cited by 86
Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells
Svenningsen, M. S., Veress, A., Harms, A., Mitarai, N., & Semsey, S. (2019) · Scientific Reports · doi:10.1038/s41598-019-42403-7 · Cited by 86
Genetically modified pigs are protected from classical swine fever virus
Xie, Z., Pang, D., Yuan, H., Jiao, H., Lu, C., Wang, K., Yang, Q., Li, M., Chen, X., Yu, T., et al. (2018) · PLoS Pathogens · doi:10.1371/journal.ppat.1007193 · Cited by 86
Proximity labeling: an emerging tool for probing in planta molecular interactions
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A technological and regulatory outlook on CRISPR crop editing
Globus, R., & Qimron, U. (2017) · Journal of Cellular Biochemistry · doi:10.1002/jcb.26303 · Cited by 89
Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System
Qazi, S., Miettinen, H. M., Wilkinson, R. A., McCoy, K., Douglas, T., & Wiedenheft, B. (2016) · Molecular Pharmaceutics · doi:10.1021/acs.molpharmaceut.5b00822 · Cited by 87
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“…the catalytically inactive or dead Cas9 (dCas9) to a variety of effector protein domains, the modulation of transcription (Gilbert et al., 2013; Qi et al., 2013), DNA methylation (McDonald et al., 2016; Vojta et al., 2016), and histone modifications (Hilton et al., 2015) have all been…”
BackgroundHallmarks of cancer: The CRISPR generation
Moses, C., García‐Bloj, B., Harvey, A. R., & Blancafort, P. (2018) · European Journal of Cancer · doi:10.1016/j.ejca.2018.01.002 · Cited by 90
DNA Methylation and Psychiatric Disorders
Liu, C., Jiao, C., Wang, K., & Yuan, N. (2018) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2018.01.006 · Cited by 86
CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity
Tsui, C. K., Barfield, R. M., Fischer, C. R., Morgens, D. W., Li, A., Smith, B. A. H., Gray, M. A., Bertozzi, C. R., Rabuka, D., & Bassik, M. C. (2019) · Nature Chemical Biology · doi:10.1038/s41589-019-0342-2 · Cited by 85
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The therapeutic application of CRISPR/Cas9 technologies for HIV
Saayman, S. M., Ali, S. A., Morris, K. V., & Weinberg, M. S. (2015) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.2015.1036736 · Cited by 91
“dCas9 has been successfully fused to accessory proteins, such as transcription activator and repressor domains, to provide RNA-guided locus-specific targeted interactions [12-17].”
BackgroundZiller, M. J., Ortega, J. A., Quinlan, K. A., Santos, D. P., Gu, H., Martin, E. J., Galonska, C., Pop, R., Maidl, S., Pardo, A. D., et al. (2018) · Cell stem cell · doi:10.1016/j.stem.2018.02.012 · Cited by 85
Bacterial CRISPR: accomplishments and prospects
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“The repression is highly specific, as determined by RNA-seq [13 ].”
InfluentialSelf-citationMature T cell responses are controlled by microRNA-142
Sun, Y., Oravecz-Wilson, K. I., Mathewson, N. D., Wang, Y., McEachin, R. C., Liu, C., Toubai, T., Wu, J. J., Rossi, C., Braun, T. W., et al. (2015) · Journal of Clinical Investigation · doi:10.1172/jci78753 · Cited by 84
CRISPR: gene editing is just the beginning
Ledford, H. K. (2016) · Nature · doi:10.1038/531156a · Cited by 84
CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects
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“To overcome this problem, knocking out or transiently silencing critical components of the NHEJ pathway could enhance the efficiency of HDR (Qi et al. 2013b).”
Genome editing in crop improvement: Present scenario and future prospects
Miglani, G. S. (2017) · Journal of Crop Improvement · doi:10.1080/15427528.2017.1333192 · Cited by 86
Quorum Sensing and Metabolic State of the Host Control Lysogeny-Lysis Switch of Bacteriophage T1
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“In order to deplete pir mRNA, cells were cotransformed with two plasmids encoding the anhydrotetracycline (aTc)-inducible dCas9 protein (a mutation of Cas9 without endonuclease activity) and constitutively expressed single guide RNA (sgRNA) targeting the coding region of pir.”
BackgroundMethodInfluentialWu, Q., Guo, Y., Lu, Y., Li, J., Guo, Y., & Jia, Z. (2020) · Genome biology · doi:10.1186/s13059-020-01984-7 · Cited by 84
“We used a well-established method to block CBS functions by dCas9 [53] [54] [55] .”
MethodOn-target activity predictions enable improved CRISPR–dCas9 screens in bacteria
Calvo‐Villamañán, A., Wong-Ng, J., Planel, R., Ménager, H., Chen, A., Cui, L., & Bikard, D. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa294 · Cited by 84
“In particular, it is well documented that guides in both orientations can effectively silence genes when binding the promoter region (1,2).”
BackgroundMethodResultInfluentialCRISPR-Cas12-based nucleic acids detection systems
Leung, R. K., Cheng, Q., Wu, Z., Khan, G., Liu, Y., Xia, H., & Wang, J. (2021) · Methods · doi:10.1016/j.ymeth.2021.02.018 · Cited by 84
A bistable genetic switch based on designable DNA-binding domains
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RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?
Unniyampurath, U., Pilankatta, R., & Krishnan, M. N. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17030291 · Cited by 88
“Furthermore, CRISPRi and CRISPRa design requires proper information on the transcription start site of the target gene [35,37,40,60].”
Backgroundcis–trans Engineering: Advances and Perspectives on Customized Transcriptional Regulation in Plants
Shrestha, A., Khan, A., & Dey, N. (2018) · Molecular Plant · doi:10.1016/j.molp.2018.05.008 · Cited by 86
CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication
Li, H., Wang, S., Dong, X., Li, Q., Li, M., Li, J., Guo, Y., Jin, X., Zhou, Y., Song, H., et al. (2020) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2020.01.028 · Cited by 84
Chen, P., Zhou, J., Wan, Y., Liu, H., Li, Y., Liu, Z., Wang, H., Lei, J., Zhao, K., Zhang, Y., et al. (2020) · Genome biology · doi:10.1186/s13059-020-01989-2 · Cited by 86
Tian, J., Yang, G., Gu, Y., Sun, X.-Q., Lü, Y., & Jiang, W. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa602 · Cited by 83
“Importantly, the gene repression strength can be optimized by adjusting the expression of dCas9 and single guide RNA (sgRNA), or by simply changing the targeting position of the sgRNA (28,31).”
El‐Sayed, A. S. A., Abdel‐Ghany, S. E., & Ali, G. S. (2017) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-017-8263-z · Cited by 86
Cai, P., Otten, A. B. C., Cheng, B., Ishii, M. A., Zhang, W., Huang, B., Qu, K., & Sun, B. K. (2019) · Genome Research · doi:10.1101/gr.251561.119 · Cited by 83
“In a classic version of CRISPRi, a catalytically dead (d) Cas9 protein is fused to a KRAB transcriptional repressor domain, which is guided to its genomic target by a single guide RNA (sgRNA) (Qi et al. 2013).”
BackgroundThe centrality of RNA for engineering gene expression
Chappell, J., Takahashi, M. K., Meyer, S., Loughrey, D., Watters, K. E., & Lucks, J. B. (2013) · Biotechnology Journal · doi:10.1002/biot.201300018 · Cited by 86
State of the art technologies to explore long non‐coding RNAs in cancer
Salehi, S., Taheri, M. N., Azarpira, N., Zare, A., & Behzad‐Behbahani, A. (2017) · Journal of Cellular and Molecular Medicine · doi:10.1111/jcmm.13238 · Cited by 84
“[84] In addition, simultaneous silencing of several genes can be brought about by utilizing several scRNAs consisting of com modules and gRNAs targeting different loci, therefore,”
BackgroundInfluentialEngineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli
Zhang, X., Guo, Y., Liu, X., Chen, X.-G., Wu, Q., & Chen, G. (2017) · Metabolic Engineering · doi:10.1016/j.ymben.2017.11.010 · Cited by 82
Progress and Challenges for Live-Cell Imaging of Genomic Loci using CRISPR-Based Platforms
Wu, X., Mao, S., Ying, Y., Krueger, C. J., & Chen, A. K. (2019) · Genomics Proteomics & Bioinformatics · doi:10.1016/j.gpb.2018.10.001 · Cited by 84
“The catalytic domains of Cas9 can be mutated to create a nuclease-deactivated form of Cas9 (dCas9), which retains the ability to interact with sgRNA and to bind to target DNA [24].”
BackgroundEndogenous Fluorescence Tagging by CRISPR
Bukhari, H., & Müller, T. (2019) · Trends in Cell Biology · doi:10.1016/j.tcb.2019.08.004 · Cited by 82
CRISPR Tools To Control Gene Expression in Bacteria
Vigouroux, A., & Bikard, D. (2020) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.00077-19 · Cited by 84
“Targeting within the promoter or in a small sequence window downstream shows strong repression and likely inhibits transcription initiation by either blocking access to the promoter or interfering with the transition of the RNAP from its initiation state into a processive elongation complex.”
BackgroundMethodInfluentialdCas9-based gene editing for cleavage-free genomic knock-in of long sequences
Wang, C., Qu, Y., Cheng, J. K. W., Hughes, N. W., Zhang, Q., Wang, M., & Cong, L. (2022) · Nature Cell Biology · doi:10.1038/s41556-021-00836-1 · Cited by 82
Lopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82
“These tools, using dCas9 fused with epigenetic modi fi ers, can modulate endogenous gene expression (Qi et al., 2013; Chavez et al., 2015; Hilton et al., 2015; Thakore et al., 2015; Liu et al., 2016) and they have been employed successfully in vivo to treat diseases such as diabetes mellitus,…”
MethodPlant–pathogen interactions: toward development of next-generation disease-resistant plants
Nejat, N., Rookes, J. E., Mantri, N. L., & Cahill, D. M. (2016) · Critical Reviews in Biotechnology · doi:10.3109/07388551.2015.1134437 · Cited by 86
Zhou, J., Wu, R., Xue, X., & Qin, Z. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw475 · Cited by 82
Liu, W., Zhang, B., & Jiang, R. (2017) · Biotechnology for Biofuels · doi:10.1186/s13068-017-0726-z · Cited by 82
“Media and growth conditions Escherichia coli DH5α was used for cloning and cultured in Luria–Bertani [20] broth with 100 μg/mL ampicillin at 37 °C.”
MethodGene Editing With CRISPR/Cas9 RNA-Directed Nuclease
Doetschman, T., & Georgieva, T. G. (2017) · Circulation Research · doi:10.1161/circresaha.116.309727 · Cited by 91
Efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli
Wu, J., Zhou, P., Zhang, X., & Dong, M. (2017) · Journal of Industrial Microbiology & Biotechnology · doi:10.1007/s10295-017-1937-9 · Cited by 82
Kinetochore inactivation by expression of a repressive mRNA
Chen, J., Tresenrider, A. M., Chia, M., McSwiggen, D. T., Spedale, G., Jorgensen, V., Liao, H., van Werven, F. J., & Ünal, E. (2017) · eLife · doi:10.7554/elife.27417 · Cited by 76
“Cell death was also rescued by silencing the pGAL-induced NDC80luti expression using CRISPRi (Qi et al., 2013) (Figure 8D), presumably due to the activation of the NDC80ORF promoter in the absence of NDC80luti transcription.”
MethodTranscriptional enhancers and their communication with gene promoters
Ray-Jones, H., & Spivakov, M. (2021) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-021-03903-w · Cited by 81
Exciting Prospects for Precise Engineering of Caenorhabditis elegans Genomes with CRISPR/Cas9
Frøkjær‐Jensen, C. (2013) · Genetics · doi:10.1534/genetics.113.156521 · Cited by 79
The state of autotrophic ethanol production in Cyanobacteria
Dexter, J., Armshaw, P., Sheahan, C., & Pembroke, J. T. (2015) · Journal of Applied Microbiology · doi:10.1111/jam.12821 · Cited by 83
“This is especially true of development of techniques that would facilitate rapid generation of genome-scale gene modulation variants and screening of resultant libraries, such as recently described CRISPRbased technologies (Bikard et al. 2013; Qi et al. 2013; Yu et al. 2013).”
BackgroundHijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects
Mougiakos, I., Bosma, E. F., Ganguly, J., John van der, O. O. S. T., & van Kranenburg, R. (2018) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2018.01.002 · Cited by 83
“…in eukaryotic systems siRNA-techniques have since long enabled transcriptional control, for prokaryotes such silencing tools have only recently become available with the CRISPR interference (CRISPRi) tool, which is based on dCas9: the catalytically inactive variant of the Cas9 endonuclease [38].”
MethodScaling up genetic circuit design for cellular computing: advances and prospects
Xiang, Y., Dalchau, N., & Wang, B. (2018) · Natural Computing · doi:10.1007/s11047-018-9715-9 · Cited by 81
A benchmark of algorithms for the analysis of pooled CRISPR screens
Bodapati, S., Daley, T., Lin, X., Zou, J., & Qi, L. S. (2020) · Genome biology · doi:10.1186/s13059-020-01972-x · Cited by 81
Self-citationBacterial CRISPR screens for gene function
Todor, H., Silvis, M. R., Osadnik, H., & Gross, C. A. (2020) · Current Opinion in Microbiology · doi:10.1016/j.mib.2020.11.005 · Cited by 82
“First, because CRISPR activity depends on the expression of both the sgRNA and dCas9 components, it is inducible and titratable [2,14].”
BackgroundExpanding the plant genome editing toolbox with recently developed CRISPR–Cas systems
Wada, N., Osakabe, K., & Osakabe, Y. (2022) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiac027 · Cited by 83
“trol (Qi et al., 2013), epigenetic editing (Gjaltema and Rots,”
Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens
Kim, S. K., Kim, H., Ahn, W., Park, K., Woo, E., Lee, D., & Lee, S. (2017) · ACS Synthetic Biology · doi:10.1021/acssynbio.6b00368 · Cited by 81
Genetic and epigenetic control of gene expression by CRISPR–Cas systems
Lo, A., & Qi, L. (2017) · F1000Research · doi:10.12688/f1000research.11113.1 · Cited by 72
Self-citationD, N. A. M., & DiFiglia, M. (2014) · Movement Disorders · doi:10.1002/mds.26020 · Cited by 83
Imaging Specific Genomic DNA in Living Cells
Chen, B., Guan, J., & Huang, B. (2016) · Annual Review of Biophysics · doi:10.1146/annurev-biophys-062215-010830 · Cited by 77
“By fusing regulatory domains to a catalytically deactivated Cas9 (dCas9), CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) has been developed to regulate endogenous gene expression (50, 85, 90, 112, 116).”
BackgroundOptimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo
Gandhi, S., Piacentino, M. L., Vieceli, F. M., & Bronner-Fraser, M. E. (2017) · Developmental Biology · doi:10.1016/j.ydbio.2017.08.036 · Cited by 79
Zhang, X., Liu, Y., Liu, L., Li, J., Du, G., & Chen, J. (2019) · Biotechnology Advances · doi:10.1016/j.biotechadv.2019.04.011 · Cited by 80
Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels
Depardieu, F., & Bikard, D. (2020) · Methods · doi:10.1016/j.ymeth.2019.07.024 · Cited by 86
“This is consistent with the fact that binding in that orientation only partially represses gene expression [14,15,27].”
BackgroundInfluentialAn expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida
Batianis, C., Kozaeva, E., Damalas, S. G., Martín‐Pascual, M., Volke, D. C., Nikel, P. I., & dos Santos, V. A. P. M. (2020) · Microbial Biotechnology · doi:10.1111/1751-7915.13533 · Cited by 80
“It has also been shown that the downregulation effect is the most efficient when dCas9 binds to the promoter sequence (Qi et al., 2013; Tan et al., 2018).”
BackgroundCRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs
Coelho, M. A., Braekeleer, E. D., Firth, M. A., Biśta, M., Łukasiak, S., Cuomo, M. E., & Taylor, B. (2020) · Nature Communications · doi:10.1038/s41467-020-17952-5 · Cited by 84
Recent Advances in CRISPR-Cas Technologies for Synthetic Biology
Jeong, S. H., Lee, H. J., & Lee, S. J. (2023) · The Journal of Microbiology · doi:10.1007/s12275-022-00005-5 · Cited by 88
“With the development of deactivated Cas9 (dCas9) in which the nucleolytic activity of the RuvC and HNH nuclease domains of Cas9 has been removed (Qi et al., 2013), a diagnostic method for detecting the binding of the dCas9/sgRNA complex to the target DNA has been studied.”
BackgroundMethodModular construction of mammalian gene circuits using TALE transcriptional repressors
Li, Y., Jiang, Y., Chen, H., Liao, W., Li, Z., Weiss, R., & Xie, Z. (2015) · Nature Chemical Biology · doi:10.1038/nchembio.1736 · Cited by 78
Design, execution, and analysis of pooled in vitro CRISPR /Cas9 screens
Miles, L. A., Garippa, R. J., & Poirier, J. T. (2016) · FEBS Journal · doi:10.1111/febs.13770 · Cited by 78
Ji, H., Jiang, Z., Lu, P., Ma, L., Li, C., Pan, H., Fu, Z., Qu, X., Wang, P., Deng, J., et al. (2016) · Molecular Therapy · doi:10.1038/mt.2016.7 · Cited by 80
CRISPR system in filamentous fungi: Current achievements and future directions
Deng, H., Gao, R., Liao, X., & Cai, Y. (2017) · Gene · doi:10.1016/j.gene.2017.06.019 · Cited by 81
L1 retrotransposition in the soma: a field jumping ahead
Faulkner, G. J., & Billon, V. (2018) · Mobile DNA · doi:10.1186/s13100-018-0128-1 · Cited by 85
An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae
Teh, M. Y., Ooi, K. H., Teo, S. X. D., Mansoor, M. E. B., Lim, W. Z. S., & Tan, M. H. (2019) · ACS Synthetic Biology · doi:10.1021/acssynbio.8b00168 · Cited by 79
Self-citationApplication of the CRISPR/Cas system for genome editing in microalgae
Zhang, Y., Jiang, J., Shi, T., Sun, X., Zhao, Q., Huang, H., & Ren, L. (2019) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-019-09726-x · Cited by 82
Truong, 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
“This system was repurposed for CRISPR interference (CRISPRi) by using a catalytically inactive Cas9 (dCas9), which orchestrates with sgRNA to sterically block the transcription of target genes (8).”
MethodMulti-input chemical control of protein dimerization for programming graded cellular responses
Wink, G., Wang, Z., Wei, C. T., Greisen, P. J., Warner, K., Cunningham‐Bryant, D., Park, K., Brunette, T., Sheffler, W., Baker, D. A., et al. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0242-8 · Cited by 79
Transcriptional control by enhancers and enhancer RNAs
Lewis, M. W., Shen, L., & Franco, H. L. (2019) · Transcription · doi:10.1080/21541264.2019.1695492 · Cited by 84
“[175] Qi LS, Larson M, Gilbert L, et al.”
BackgroundMouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells
Qizhao, W., Zhuang, P., Huang, H., Li, L., Liu, L., Webber, H. C., Dalal, R., Siew, L., Fligor, C. M., Chang, K., et al. (2020) · Journal of Neuroscience · doi:10.1523/jneurosci.0102-20.2020 · Cited by 82
“RNA-guided endonuclease Cas9 has been repurposed for transcriptional inhibition and activation using catalytically dead Cas9 (dCas9; Qi et al., 2013; Gilbert et al., 2014; La Russa and Qi, 2015; Gao et al., 2016; Chen and Qi, 2017; Joung et al., 2017), but the much larger size of transcriptional…”
MethodThermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging
Knutson, S. D., Sanford, A. A., Swenson, C., Korn, M. M., Manuel, B. A., & Heemstra, J. M. (2020) · Journal of the American Chemical Society · doi:10.1021/jacs.0c08996 · Cited by 79
CRISPR Interference To Inducibly Repress Gene Expression in Chlamydia trachomatis
Ouellette, S. P., Blay, E. A., Hatch, N. D., & Fisher, L. A. (2021) · Infection and Immunity · doi:10.1128/iai.00108-21 · Cited by 79
“In Escherichia coli, these components are typically encoded on two plasmids (17), but for Chlamydia, we created a single-plasmid system (16).”
BackgroundCurrent Techniques to Study Beneficial Plant-Microbe Interactions
Gamalero, E., Bona, E., & Glick, B. R. (2022) · Microorganisms · doi:10.3390/microorganisms10071380 · Cited by 80
Engineering approaches for RNA-based and cell-based osteoarthritis therapies
DeJulius, C. R., Walton, B. L., Colazo, J. M., d’Arcy, R., Francini, N., Brunger, J. M., & Duvall, C. L. (2024) · Nature Reviews Rheumatology · doi:10.1038/s41584-023-01067-4 · Cited by 85
“with a repressor domain, typically the KRAB domain from human zinc finger KOX1 (dCas9–KRAB) 170–172 .”
Heman-Ackah, S. M., Bassett, A. R., & Wood, M. J. A. (2016) · Scientific Reports · doi:10.1038/srep28420 · Cited by 77
“Since the discovery of this system in 2013 37 , coupling of dCas9 to repressive domains has been used to enhance CRISPRi in eukaryotes 38 .”
InfluentialCasFinder: Flexible algorithm for identifying specific Cas9 targets in genomesPreprint
Aach, J., Mali, P., & Church, G. M. (2014) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/005074 · Cited by 80
A simple, flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system
Kim, H., Kim, S., Ryu, J., Choi, M. K., Kweon, J., Kang, B., Ahn, H., Bae, S., Kim, J., Kim, J., et al. (2016) · Journal of Integrative Plant Biology · doi:10.1111/jipb.12474 · Cited by 78
““Dead” Cas9, lacking both nuclease active sites with only target recognition, has been used to perform chromatin immunoprecipitation, targeted gene activation/ repression, or more specific genome editing (Qi et al. 2013; Guilinger et al. 2014).”
MethodLainšček, D., Kadunc, L., Keber, M. M., Hafner‐Bratkovič, I., Romih, R., & Jerala, R. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.8b00192 · Cited by 78
Placental imprinting: Emerging mechanisms and functions
Hanna, C. W. (2020) · PLoS Genetics · doi:10.1371/journal.pgen.1008709 · Cited by 78
Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing
Lyu, P., Wang, L., & Lu, B. (2020) · Life · doi:10.3390/life10120366 · Cited by 79
CRISPR Therapeutics for Duchenne Muscular Dystrophy
Erkut, E. J. N., & Yokota, T. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23031832 · Cited by 82
“Through a dCas9-transcriptional activator fusion protein, the UTRN promoters can be targeted thus increasing expression (Figure 4B) [49].”
BackgroundCRISPR/Cas Systems‐Inspired Nano/Biosensors for Detecting Infectious Viruses and Pathogenic Bacteria
Zhang, X., Shi, Y., Chen, G., Wu, D., Wu, Y., & Li, G. (2022) · Small Methods · doi:10.1002/smtd.202200794 · Cited by 83
Effects of Genetic Variation on the E. coli Host-Circuit Interface
Cardinale, S., Joachimiak, M. P., & Arkin, A. P. (2013) · Cell Reports · doi:10.1016/j.celrep.2013.06.023 · Cited by 78
Self-citationReassessing Escherichia coli as a cell factory for biofuel production
Wang, C., Pfleger, B. F., & Kim, S.-W. (2017) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2017.02.010 · Cited by 78
Goyal, A., Fiškin, E., Gutschner, T., Polycarpou‐Schwarz, M., Groß, M., Neugebauer, J. K., Gandhi, M., Caudron‐Herger, M., Beneš, V., & Diederichs, S. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx952 · Cited by 77
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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