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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
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
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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×
“The CRISPR (clustered regularly interspaced short palindromic repeats) system provides a potential platform for targeted gene regulation (Barrangou et al., 2007).”
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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).”
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Human myotube formation is determined by MyoD–Myomixer/Myomaker axis
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Integration of CRISPR/Cas9 with artificial intelligence for improved cancer therapeutics
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“developed the catalytically dead dCas9 protein, deficient in endonuclease activity but able to initiate CRISPR interference (CRISPRi) and repress target genes [25].”
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Prospects of microbial cell factories developed through systems metabolic engineering
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“For this, various methods including transcription factor engineering (Alper et al., 2006), and gene knockdown libraries based on sRNA (Na et al., 2013) or CRISPR interference (CRISPRi; Qi et al., 2013) can be employed.”
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Zhou, S., Ding, R., Chen, J., Du, G., Li, H., & Zhou, J. (2017) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00006 · Cited by 100
Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9
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CRISPR-based bioengineering in microalgae for production of industrially important biomolecules
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“coli [33 ,34], S.”
Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii
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“Novel global genomic approaches, such as CRISPR interference (CRISPRi), promise to accelerate the understanding of gene function and regulation [124,131].”
BackgroundAdvancing chimeric antigen receptor T cell therapy with CRISPR/Cas9
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“…et al., 2013; Gilbert et al., 2013; Hwang et al., 2013; Li et al., 2013; Maeder et al., 2013; Nekrasov et al., 2013; Perez-Pinera et al., 2013; Qi et al., 2013; Shen et al., 2013; Wang et al., 2013; Tanenbaum et al., 2014; Chavez et al., 2015; Hilton et al., 2015; Kearns et al., 2015;…”
BackgroundInsulated transcriptional elements enable precise design of genetic circuits
Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q., & Lou, C. (2017) · Nature Communications · doi:10.1038/s41467-017-00063-z · Cited by 101
Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids
Fang, L., Fan, J., Luo, S., Chen, Y., Wang, C., Cao, Y., & Song, H. (2021) · Nature Communications · doi:10.1038/s41467-021-25243-w · Cited by 99
Tremblay, F., Xiong, Q., Shah, S. S., Ko, C., Kelly, K., Morrison, M. S., Giancarlo, C., Ramírez, R. N., Hildebrand, E. M., Voytek, S. B., et al. (2025) · Nature Medicine · doi:10.1038/s41591-025-03508-x · Cited by 116
Chung, J. Y., Ain, Q. U., Song, Y., Yong, S., & Kim, Y. (2019) · Genome Research · doi:10.1101/gr.246900.118 · Cited by 102
“Several types of genome editing using the CRISPR system and genome modification using a catalytically inactive version of Cas9 for RNA-guided transcription regulations, known as CRISPR interference (CRISPRi), have been studied (Qi et al. 2013).”
BackgroundWong, G. C. L., Antani, J. D., Lele, P. P., Chen, J., Nan, B., Kühn, M. J., Persat, A., Bru, J.-L., Høyland‐Kroghsbo, N. M., Siryaporn, A., et al. (2021) · Physical Biology · doi:10.1088/1478-3975/abdc0e · Cited by 93
“Combining such techniques with dynamic gene perturbation methods [10] is anticipated to reveal bacterial adaptations that may occur following surface adhesion; these are likely to be missed in current approaches that tend to focus on the steady-state responses to the loss of enzymatic function.”
BackgroundXu, L., Zhao, L., Gao, Y., Xu, J., & Han, R. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw1048 · Cited by 91
New variants of CRISPR RNA‐guided genome editing enzymes
Murovec, J., Pirc, Ž., & Yang, B. (2017) · Plant Biotechnology Journal · doi:10.1111/pbi.12736 · Cited by 102
“Nowadays, CRISPR/Cas9 technology can also enable spatiotemporal modulation of gene transcript levels using dSpCas9 alone (so-called CRISPR interference or CRISPRi technology; Qi et al., 2013) or fused with appropriate gene transcription activation or repression domains (Perez-Pinera et al.”
MethodCRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces
Zhao, Y., Li, L., Zheng, G., Jiang, W., Deng, Z., Wang, Z., & Lü, Y. (2018) · Biotechnology Journal · doi:10.1002/biot.201800121 · Cited by 98
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CRISPR/Cas System and Factors Affecting Its Precision and Efficiency
Javaid, N., & Choi, S. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.761709 · Cited by 106
“Transcriptional inhibition by the CRISPR machinery is termed CRISPRi and only dcas9 is sufficient in bacterial cells via steric hindrance of the transcriptional machinery (Bikard et al., 2013; Qi et al., 2013).”
BackgroundJoly, J. H., Delfarah, A., Phung, P. S., Parrish, S., & Graham, N. A. (2020) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)49891-7 · Cited by 97
Inhibitors of bacterial immune systems: discovery, mechanisms and applications
Mayo-Muñoz, D., Pinilla‐Redondo, R., Camara-Wilpert, S., Birkholz, N., & Fineran, P. C. (2024) · Nature Reviews Genetics · doi:10.1038/s41576-023-00676-9 · Cited by 99
CRISPR-Cas systems: new players in gene regulation and bacterial physiology
Sampson, T. R., & Weiss, D. S. (2014) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2014.00037 · Cited by 98
Synthetic analog and digital circuits for cellular computation and memory
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Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system
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Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases
Standage-Beier, K., Zhang, Q., & Wang, X. (2015) · ACS Synthetic Biology · doi:10.1021/acssynbio.5b00132 · Cited by 96
Gao, C., Wang, S., Hu, G., Guo, L., Chen, X., Xu, P., & Liu, L. (2017) · Biotechnology and Bioengineering · doi:10.1002/bit.26486 · Cited by 96
“To reconcile the huge disparity in protein expression levels in vivo, CRISPRi technology was employed owing to its features of simple manipulation in multiplexed transcriptional regulation with order-of-magnitude dynamic range (Dominguez et al. 2016; Qi et al. 2013).”
BackgroundMethodWang, Q., & Coleman, J. J. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.06.007 · Cited by 103
“In human cells, this interference efficiency can be up to 90% [51].”
BackgroundBaumgarten, S., Bryant, J. M., Sinha, A., Reyser, T., Preiser, P. R., Dedon, P. C., & Scherf, A. (2019) · Nature Microbiology · doi:10.1038/s41564-019-0521-7 · Cited by 95
Multiplexed genome regulation in vivo with hyper-efficient Cas12a
Guo, L. Y., Bian, J. T., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., et al. (2022) · Nature Cell Biology · doi:10.1038/s41556-022-00870-7 · Cited by 100
Self-citationCas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity
Rostain, W., Grebert, T., Vyhovskyi, D., Pizarro, P. T., Tshinsele-Van Bellingen, G., Cui, L., & Bikard, D. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad170 · Cited by 85
“dCas9 can efficiently block transcription initiation when binding within promoter sequences or transcription elongation when binding downstream of promoters (7,8).”
BackgroundSingle-cell and multivariate approaches in genetic perturbation screens
Liberali, P., Snijder, B., & Pelkmans, L. (2014) · Nature Reviews Genetics · doi:10.1038/nrg3768 · Cited by 94
CRISPR-mediated genome editing and human diseases
Cai, L., Fisher, A. L., Huang, H., & Xie, Z. (2016) · Genes & Diseases · doi:10.1016/j.gendis.2016.07.003 · Cited by 83
“Alternately, a nuclease-deficient Cas9, called “dead Cas9” or dCas9 was developed by Qi et al.(21) The dCas9 protein has no DNA cleavage activity and has been used to create fusion proteins that target either the promoter or other regulatory sequence of a gene with the goal of altering gene expression.”
BackgroundEnhanced protein and biochemical production using CRISPRi-based growth switches
Li, S., Jendresen, C. B., Grünberger, A., Ronda, C., Jensen, S. I., Noack, S., & Nielsen, A. T. (2016) · Metabolic Engineering · doi:10.1016/j.ymben.2016.09.003 · Cited by 95
Zuberi, A., Misba, L., & Khan, A. U. (2017) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2017.00214 · Cited by 95
“The PCR products were purified, ligated and transformed in TOP 10, competent E. coli cells and the data was confirmed through sequencing of linearized DNA fragments containing target specific sgRNA, generated through colony PCR with different set of primers (Supplementary Table 2).”
BackgroundInfluentialEngineering Diagnostic and Therapeutic Gut Bacteria
Landry, B. P., & Tabor, J. J. (2017) · Microbiology Spectrum · doi:10.1128/microbiolspec.bad-0020-2017 · Cited by 95
CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification
Oakes, B. L., Fellmann, C., Rishi, H. S., Taylor, K. L., Ren, S. M., Nadler, D. C., Yokoo, R., Arkin, A. P., Doudna, J. A., & Savage, D. F. (2019) · Cell · doi:10.1016/j.cell.2018.11.052 · Cited by 94
“Plasmids used were based on a 2 plasmid system as reported previously (Oakes et al., 2014, 2016; Qi et al., 2013) containing Cas9 and variants on a selectable CmR marker and plasmids with sgRNAs and proteases with AmpR markers, representative sequences of which can be found in Table S1.”
InfluentialSelf-citationTadić, V., Josipović, G., Zoldoš, V., & Vojta, A. (2019) · Methods · doi:10.1016/j.ymeth.2019.05.003 · Cited by 99
Zhang, S., Guo, F., Yan, W., Dai, Z., Dong, W., Zhou, J., Zhang, W., Xin, F., & Jiang, M. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2019.00459 · Cited by 95
Newsom, S. N., Parameshwaran, H. P., Martin, L., & Rajan, R. (2021) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2020.619763 · Cited by 99
“Once CASCADE locates the target DNA, Cas3 is recruited for DNA cleavage (Sinkunas et al., 2011).”
BackgroundInfluentialCao, X., Li, B., Chen, J., Dang, J., Chen, S., Gunes, E. G., Xu, B., Tian, L., Muend, S., Raoof, M., et al. (2021) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2020-002022 · Cited by 97
CRISPR screens for functional interrogation of immunity
Shi, H., Doench, J. G., & Chi, H. (2022) · Nature reviews. Immunology · doi:10.1038/s41577-022-00802-4 · Cited by 102
A versatile framework for microbial engineering using synthetic non-coding RNAs
Qi, L. S., & Arkin, A. P. (2014) · Nature Reviews Microbiology · doi:10.1038/nrmicro3244 · Cited by 96
Self-citationGenome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System
Wang, S., Dong, S., Wang, P., Tao, Y., & Wang, Y. (2017) · Applied and Environmental Microbiology · doi:10.1128/aem.00233-17 · Cited by 97
“As shown in Table 1, by following the same procedure to obtain the pta deletion mutant, it was determined that the order of the promoters for efficient gRNA expression (and thus genome editing efficiency) is PJ23119 Pvegb PsRNA Pvegc.”
MethodInfluentialPICKLES: the database of pooled in-vitro CRISPR knockout library essentiality screens
Lenoir, W. F., Lim, T. L., & Hart, T. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx993 · Cited by 94
“The most commonly used CRISPR-associated endonuclease, SpCas9, has been modified to disable its endonuclease activity, facilitating protein fusion with domains for transcriptional activation (13,14), transcriptional repression (13,15), and chromatin modification (16).”
MethodA sigma factor toolbox for orthogonal gene expression in Escherichia coli
Bervoets, I., Brempt, M. V., Nerom, K. V., Hove, B. V., Maertens, J., Mey, M. D., & Charlier, D. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky010 · Cited by 100
Schwartz, C., Curtis, N. C., Löbs, A., & Wheeldon, I. R. (2018) · Biotechnology Journal · doi:10.1002/biot.201700584 · Cited by 94
CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium
Sekine, R., Kawata, T., & Muramoto, T. (2018) · Scientific Reports · doi:10.1038/s41598-018-26756-z · Cited by 89
Dong, H., Xiang, H., Mu, D., Wang, D., & Wang, T. (2018) · International Journal of Antimicrobial Agents · doi:10.1016/j.ijantimicag.2018.09.017 · Cited by 94
Improved sgRNA design in bacteria via genome-wide activity profiling
Guo, J., Wang, T., Guan, C., Liu, B., Luo, C. Y., Xie, Z., Zhang, C., & Xing, X. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky572 · Cited by 99
Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects
Zhang, R., Xu, W., Shao, S., & Wang, Q. (2021) · Frontiers in Microbiology · doi:10.3389/fmicb.2021.635227 · Cited by 96
“The function of dCas9 was confirmed by the native elongating transcript sequencing (NETseq) experiment (Qi et al., 2013) and Figure 1 demonstrated the RNAP is blocked by the dCas9-sgRNA complexes.”
BackgroundInfluentialSynthetic Biology in Natural Product Biosynthesis
Seshadri, K., Abad, A. N. D., Nagasawa, K. K., Yost, K. M., Johnson, C. W., Dror, M. J., & Tang, Y. (2025) · Chemical Reviews · doi:10.1021/acs.chemrev.4c00567 · Cited by 94
Cyanobacterial chassis engineering for enhancing production of biofuels and chemicals
Gao, X., Sun, T., Pei, G., Chen, L., & Zhang, W. (2016) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-016-7374-2 · Cited by 106
Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras
Shis, D. L., Hussain, F., Meinhardt, S., Swint‐Kruse, L., & Bennett, M. R. (2014) · ACS Synthetic Biology · doi:10.1021/sb500262f · Cited by 93
CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing
Noman, A., Aqeel, M. A., & He, S. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.01740 · Cited by 97
Lu, B., Javidi-Parsijani, P., Makani, V. S., Mehraein‐Ghomi, F., Sarhan, W. M., Sun, D., Yoo, K. W., Atala, Z., Lyu, P., & Atala, A. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz093 · Cited by 94
CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris
Gassler, T., Heistinger, L., Mattanovich, D., Gasser, B., & Prielhofer, R. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-9024-5_9 · Cited by 92
Meyer, M. B., & Pike, J. W. (2019) · The Journal of Steroid Biochemistry and Molecular Biology · doi:10.1016/j.jsbmb.2019.105500 · Cited by 93
Improvements in algal lipid production: a systems biology and gene editing approach
Banerjee, A., Banerjee, C., Negi, S., Chang, J., & Shukla, P. (2017) · Critical Reviews in Biotechnology · doi:10.1080/07388551.2017.1356803 · Cited by 95
Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering
Nuñez, J. K., Harrington, L. B., & Doudna, J. A. (2016) · ACS Chemical Biology · doi:10.1021/acschembio.5b01019 · Cited by 92
Self-citationSingh, V., Gohil, N., Ramirez-Garcia, R., Braddick, D., & Fofié, C. K. (2017) · Journal of Cellular Biochemistry · doi:10.1002/jcb.26165 · Cited by 92
“This construct is used for gene repression, activation, loci imaging, and epigenetic studies [Bikard et al., 2013; Chen et al., 2013; Gilbert et al., 2013; Qi et al., 2013; Ma et al., 2015].”
BackgroundMethodYoung, J. C., Dominicus, C. S., Wagener, J., Butterworth, S. W., Ye, X., Kelly, G. P., Ordan, M., Saunders, B., Instrell, R., Howell, M. T., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11855-w · Cited by 92
Cas9 Cleavage of Viral Genomes Primes the Acquisition of New Immunological Memories
Nussenzweig, P. M., McGinn, J., & Marraffini, L. A. (2019) · Cell Host & Microbe · doi:10.1016/j.chom.2019.09.002 · Cited by 89
Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering
Zheng, Y., Li, J., Wang, B., Han, J., Hao, Y., Wang, S., Ma, X., Yang, S., Ma, L., Li, Y., et al. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.00062 · Cited by 92
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox
Koonin, E. V., Gootenberg, J. S., & Abudayyeh, O. O. (2023) · Biochemistry · doi:10.1021/acs.biochem.3c00159 · Cited by 95
Carte, J. M., Christopher, R. T., Smith, J. T., Olson, S., Barrangou, R., Moineau, S., Glover, C. V. C., Graveley, B. R., Terns, R. M., & Terns, M. P. (2014) · Molecular Microbiology · doi:10.1111/mmi.12644 · Cited by 95
Tuning the dials of Synthetic Biology
Arpino, J. A. J., Hancock, E. J., Anderson, J., Barahona, M., Stan, G.-B. V., Papachristodoulou, A., & Polizzi, K. M. (2013) · Microbiology · doi:10.1099/mic.0.067975-0 · Cited by 92
Kampmann, M., Bassik, M. C., & Weissman, J. S. (2014) · Nature Protocols · doi:10.1038/nprot.2014.103 · Cited by 90
Self-citationWaking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system
García‐Bloj, B., Moses, C., Sgro, A., Plani-Lam, J. H. C., Arooj, M., Duffy, C., Thiruvengadam, S., Sorolla, A., Rashwan, R., Mancera, R. L., et al. (2016) · Oncotarget · doi:10.18632/oncotarget.11142 · Cited by 92
Liu, T., Li, Z., Zhang, Q., Bernstein, K. D. A., Lozano‐Calderón, S. A., Choy, E., Hornicek, F. J., & Duan, Z. (2016) · Oncotarget · doi:10.18632/oncotarget.13148 · Cited by 96
“CRISPR-Cas9 guided gene targeting is highly specific [33, 35].”
BackgroundMethodInfluentialTranscription control engineering and applications in synthetic biology
Engstrom, M. D., & Pfleger, B. F. (2017) · Synthetic and Systems Biotechnology · doi:10.1016/j.synbio.2017.09.003 · Cited by 96
“[268] used a dCas9 variant to block transcription initiation and elongation using sgRNAs (single guide RNAs) to various targets in the promoters and coding sequences of variant rfp and gfp reporter genes.”
MethodProgrammable CRISPR‐Cas transcriptional activation in bacteria
Ho, H., Fang, J. R., Cheung, J., & Wang, H. H. (2020) · Molecular Systems Biology · doi:10.15252/msb.20199427 · Cited by 91
“The Cas9 protein, a member of a large class of RNA-guided DNA nucleases, has emerged over the past several years as a promising system for building synthetic TFs (Bikard et al , 2013; Qi et al , 2013).”
InfluentialCRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 98
“In addition, dCas9-KRAB, stably integrated into the genome in HeLa cells, caused a stable 5–10-fold repression of genes and promoter regions [93], and a 100-fold effect was observed when the target site was located at 50–100 bp downstream of the transcription start site [90].”
BackgroundTranscriptional regulation with CRISPR-Cas9: principles, advances, and applications
Didovyk, A., Borek, B., Tsimring, L., & Hasty, J. (2016) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2016.06.003 · Cited by 90
“Despite this, a few mismatches between guide sequence and target DNA can be tolerated [5 ,7,8 ,9], more so within the 5’ proximal position of the guide sequence.”
Wu, J., Zhang, X., Xia, X., & Dong, M. (2017) · Metabolic Engineering · doi:10.1016/j.ymben.2017.03.012 · Cited by 90
Editing streptomycete genomes in the CRISPR/Cas9 age
Alberti, F., & Corre, C. (2019) · Natural Product Reports · doi:10.1039/c8np00081f · Cited by 93
Chlamydial Infection From Outside to Inside
Gitsels, A., Sanders, N. N., & Vanrompay, D. C. G. (2019) · Frontiers in Microbiology · doi:10.3389/fmicb.2019.02329 · Cited by 90
A navigation guide of synthetic biology tools for Pseudomonas putida
Martín‐Pascual, M., Batianis, C., Bruinsma, L., Asin‐Garcia, E., Morales, L. G., Weusthuis, R. A., van Kranenburg, R., & dos Santos, V. A. P. M. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107732 · Cited by 92
CRISPRi-seq for genome-wide fitness quantification in bacteria
de Bakker, V., Liu, X., Bravo, A. M., & Veening, J. (2022) · Nature Protocols · doi:10.1038/s41596-021-00639-6 · Cited by 90
“subtilis studies of Qi et al.(2) and Hawkins et al.”
BackgroundFujita, T., Asano, Y., Ohtsuka, J., Takada, Y. K., Saito, K., Ohki, R., & Fujii, H. (2013) · Scientific Reports · doi:10.1038/srep03171 · Cited by 93
CRISPR-Based Typing and Next-Generation Tracking Technologies
Barrangou, R., & Dudley, E. G. (2016) · Annual Review of Food Science and Technology · doi:10.1146/annurev-food-022814-015729 · Cited by 91
Effective gene editing by high-fidelity base editor 2 in mouse zygotes
Liang, P., Sun, H., Sun, Y., Zhang, X., Xie, X., Zhang, J., Zhang, Z., Chen, Y., Ding, C., Xiong, Y., et al. (2017) · Protein & Cell · doi:10.1007/s13238-017-0418-2 · Cited by 91
“We were surprised to find low-frequency (2/44 embryos) base insertions and deletions at target sites with HF2-BE2, even though dCas9 has no demonstrable nuclease activity (Hsu et al., 2014; Mali et al., 2013; Qi et al., 2013).”
BackgroundCao, Y., Li, X., Li, F., & Song, H. (2017) · ACS Synthetic Biology · doi:10.1021/acssynbio.6b00374 · Cited by 89
Shabestary, K., Anfelt, J., Ljungqvist, E., Jahn, M., Yao, L., & Hudson, E. P. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.8b00056 · Cited by 89
CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives
Chan, Y.-T., Lu, Y., Wu, J., Zhang, C., Tan, H., Bian, Z., Wang, N., & Feng, Y. (2022) · Theranostics · doi:10.7150/thno.71144 · Cited by 93
“The use of catalytically inactive Cas9 (dCas9) could repress target genes by transcriptional inhibition, also known as CRISPR interference (CRISPRi) [23-25].”
BackgroundSystematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells
Stanton, B. C., Siciliano, V., Ghodasara, A., Wróblewska, L., Clancy, K., Trefzer, A. C., Chesnut, J. D., Weiss, R., & Voigt, C. A. (2014) · ACS Synthetic Biology · doi:10.1021/sb5002856 · Cited by 88
In situ genotyping of a pooled strain library after characterizing complex phenotypes
Lawson, M. J., Camsund, D., Larsson, J., Baltekin, Ö., Fange, D., & Elf, J. (2017) · Molecular Systems Biology · doi:10.15252/msb.20177951 · Cited by 82
“Design and construction of the DuMPLING screening strain The dCas9 expression cassette from Qi et al (2013), which includes the TetR repressor and the bidirectional PRPA promoter that regulates both tetR and dcas9, was introduced into the chromosome of E. coli and optimized for low leakage of dCas9…”
MethodTargeted DNA methylation in human cells using engineered dCas9-methyltransferases
Xiong, T., Meister, G. E., Workman, R. E., Kato, N. C., Spellberg, M. J., Türker, F., Timp, W., Ostermeier, M., & Novina, C. D. (2017) · Scientific Reports · doi:10.1038/s41598-017-06757-0 · Cited by 88
CRISPR Approaches to Small Molecule Target Identification
Jost, M., & Weissman, J. S. (2018) · ACS Chemical Biology · doi:10.1021/acschembio.7b00965 · Cited by 88
Self-citationMolecular Toolkit for Gene Expression Control and Genome Modification inRhodococcus opacusPD630
DeLorenzo, D. M., Rottinghaus, A. G., Henson, W. R., & Moon, T. S. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00416 · Cited by 88
Programming Bacteria With Light—Sensors and Applications in Synthetic Biology
Liu, Z., Zhang, J., Jin, J., Geng, Z., Qi, Q., & Liang, Q. (2018) · Frontiers in Microbiology · doi:10.3389/fmicb.2018.02692 · Cited by 88
Squamous trans-differentiation of pancreatic cancer cells promotes stromal inflammation
Somerville, T. D. D., Biffi, G., Daßler‐Plenker, J., Hur, S. K., He, X., Vance, K. E., Miyabayashi, K., Xu, Y., Maia-Silva, D., Klingbeil, O., et al. (2020) · eLife · doi:10.7554/elife.53381 · Cited by 87
“A major cell type in the stroma of PDA tumors are CAFs, which have historically been considered tumor-promoting, however recent studies suggest they may also have tumor restraining functions (Öhlund et al., 2014; Özdemir et al., 2014; Rhim et al., 2014).”
BackgroundCRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes
Tong, Y., Whitford, C. M., Blin, K., Jørgensen, T. S., Weber, T., & Lee, S. Y. (2020) · Nature Protocols · doi:10.1038/s41596-020-0339-z · Cited by 85
Illuminating the noncoding genome in cancer
Zhang, X., & Meyerson, M. M. (2020) · Nature Cancer · doi:10.1038/s43018-020-00114-3 · Cited by 86
Donati, S., Kuntz, M. A., Pahl, V., Farke, N., Beuter, D., Glatter, T., Gomes‐Filho, J. V., Randau, L., Wang, C., & Link, H. (2020) · Cell Systems · doi:10.1016/j.cels.2020.10.011 · Cited by 83
“, 2017), and a sgRNA on a plasmid (Qi et al., 2013).”
BackgroundInfluentialGenome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs
Brunger, J. M., Zutshi, A., Willard, V. P., Gersbach, C. A., & Guilak, F. (2017) · Stem Cell Reports · doi:10.1016/j.stemcr.2017.03.022 · Cited by 88
“, 2011), and synthetic transcription regulators (Liu et al., 2001; Perez-Pinera et al., 2013a, 2013b; Qi et al., 2013) will facilitate the generation of complex circuits capable of integrating multi-input cues and may enable a cell to discern and respond specifically to not only varying degrees of inflammation, but al…”
Background
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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