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Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
Open accessAbstract
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.
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“Because of the relatively large size of 4277 bp of dCas9-VPR cDNA, the authors used a dual rAAV system to express the dCas9-VPR and gRNA expression cassettes [223,224].”
From Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays (2020) · cites this paper as [224]
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“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 [30]
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“Novel strategies of Cas9-based transcription modulators rapidly appeared prompted by the fusion of dCas9 or gRNA to different activator or repressor elements, with multiple degrees of modulation and specificity, and enabling single or multiplexed transcriptional control [74,75,76].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [74]
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“This dCas9-effector strategy has already been applied in genome wide screening study in which gain-of-function mutations in melanoma cells that confer drug resistance were identified.46”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [46]
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“…to target each promoter based on previous reports indicating that multiple gRNAs at a single promoter are more effective for gene activation 15,16,18,19,21,22 . dCas9-VP64 was able to induce EpCAM gene expression in both cell lines, whereas gRNA-directed dCas-PRDM9 and dCas-VP64 were ineffective…”
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“CRISPR/Cas9 has also been used to mediate efficient transcriptional activation in human cells [32].”
MethodA novel enhancer regulates MGMT expression and promotes temozolomide resistance in glioblastoma
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Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease
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InfluentialSelf-citationBlue-Light Receptors for Optogenetics
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Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
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“In addition to generating gene knockouts, the CombiGEM-CRISPR platform could be used for high-order combinatorial gene activation and repression studies by incorporating gRNAs and deactivated Cas9 variants repurposed as transcriptional and epigenetic regulators (8, 9, 51, 52).”
MethodRobust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems
Lowder, L. G., Zhou, J., Zhang, Y., Malzahn, A., Zhong, Z., Hsieh, T.-F., Voytas, D. F., Zhang, Y., & Qi, Y. (2018) · Molecular Plant · doi:10.1016/j.molp.2017.11.010 · Cited by 232
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“Using the CRISPR synergistic activation mediate (CRISPRa SAM) system (Konermann et al., 2015) (Figure 4C), which takes advantage of the DNA targeting capabilities of a catalytically dead Cas9 protein fused to VP64 and a modified sgRNA that recruits and targets transcriptional activators to promoter…”
MethodLive cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9
Qin, P., Parlak, M., Kuscu, C., Bandaria, J. N., Mir, M., Szlachta, K. A., Singh, R., Darzacq, X., Yıldız, A., & Adli, M. (2017) · Nature Communications · doi:10.1038/ncomms14725 · Cited by 238
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Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants
Kumar, M., Prusty, M. R., Pandey, M. K., Singh, P. K., Bohra, A., Guo, B. Z., & Varshney, R. K. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1157678 · Cited by 251
“In this way, the fusion protein is guided to reach specific sites in the genome to do its job (Konermann et al., 2015).”
BackgroundMethods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes
Hess, G. T., Tycko, J., Yao, D., & Bassik, M. C. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.09.029 · Cited by 234
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The New State of the Art: Cas9 for Gene Activation and Repression
Russa, M. F. L., & Qi, L. S. (2015) · Molecular and Cellular Biology · doi:10.1128/mcb.00512-15 · Cited by 241
“Recently, both CRISPRa and CRISPRi have been employed in pooled genetic screens in mammalian cells (29, 32).”
BackgroundMethodInfluentialEpitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication
Courtney, D., Kennedy, E. M., Dumm, R. E., Bogerd, H. P., Tsai, K., Heaton, N. S., & Cullen, B. R. (2017) · Cell Host & Microbe · doi:10.1016/j.chom.2017.08.004 · Cited by 229
“…RNAs results in the insertion of characteristic T to C mutations at sites of protein crosslinking to 4SU residues, thus allowing the exclusion from the subsequent bioinformatics analysis of all background reads resulting from non-crosslinked RNAs (Kennedy et al., 2016; Konermann et al., 2015).”
BackgroundMethodInfluentialCell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks
Alyami, M., Alsaiari, S. K., Li, Y., Qutub, S., Aleisa, F. A., Sougrat, R., Merzaban, J. S., & Khashab, N. M. (2020) · Journal of the American Chemical Society · doi:10.1021/jacs.9b11638 · Cited by 229
Long Non-coding RNA Structure and Function: Is There a Link?
Zampetaki, A., Albrecht, A. A., & Steinhöfel, K. K. (2018) · Frontiers in Physiology · doi:10.3389/fphys.2018.01201 · Cited by 231
Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria
Dong, C., Fontana, J. T., Patel, A., Carothers, J. M., & Zalatan, J. G. (2018) · Nature Communications · doi:10.1038/s41467-018-04901-6 · Cited by 221
Potential of Genome Editing to Improve Aquaculture Breeding and Production
Gratacap, R. L., Wargelius, A., Edvardsen, R. B., & Houston, R. D. (2019) · Trends in Genetics · doi:10.1016/j.tig.2019.06.006 · Cited by 227
A transcription factor atlas of directed differentiation
Joung, J., Ma, S., Tay, T., Geiger-Schuller, K., Kirchgatterer, P. C., Verdine, V. K., Guo, B., Arias-García, M. A., Allen, W. E., Singh, A., et al. (2023) · Cell · doi:10.1016/j.cell.2022.11.026 · Cited by 229
Self-citationA CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death
Ma, H., Dang, Y., Wu, Y., Jia, G., Anaya, E., Zhang, J., Abraham, S., Choi, J.-G., Shi, G., Qi, L., et al. (2015) · Cell Reports · doi:10.1016/j.celrep.2015.06.049 · Cited by 223
“Thus, the CRISPR system has potential as an improved approach for performing genome-wide screening, as suggested by recent reports (Gilbert et al., 2014; Koike-Yusa et al., 2014; Konermann et al., 2014; Sanjana et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014).”
BackgroundLoss-of-function genetic tools for animal models: cross-species and cross-platform differences
Housden, B. E., Muhar, M., Gemberling, M. P., Gersbach, C. A., Stainier, D. Y. R., Seydoux, G., Mohr, S. E., Zuber, J. A., & Perrimon, N. (2016) · Nature Reviews Genetics · doi:10.1038/nrg.2016.118 · Cited by 225
Identifying chemogenetic interactions from CRISPR screens with drugZ
Colic, M., Wang, G., Zimmermann, M., Mascall, K. S., McLaughlin, M. E., Bertolet, L. L., Lenoir, W. F., Moffat, J., Angers, S., Durocher, D., et al. (2019) · Genome Medicine · doi:10.1186/s13073-019-0665-3 · Cited by 211
CRISPR/Cas9 advances engineering of microbial cell factories
Jakočiūnas, T., Jensen, M. K., & Keasling, J. D. (2015) · Metabolic Engineering · doi:10.1016/j.ymben.2015.12.003 · Cited by 225
Esposito, R., Bosch, N., Lanzós, A., Polidori, T., Pulido-Quetglas, C., & Johnson, R. (2019) · Cancer Cell · doi:10.1016/j.ccell.2019.01.019 · Cited by 223
NEAT1 is essential for metabolic changes that promote breast cancer growth and metastasis
Park, M. K., Zhang, L., Min, K., Cho, J.-H., Yeh, C., Moon, H., Hormaechea‐Agulla, D., Mun, H., Ko, S., Lee, J. W., et al. (2021) · Cell Metabolism · doi:10.1016/j.cmet.2021.11.011 · Cited by 216
A Thermostable Cas9 with Increased Lifetime in Human Plasma
Harrington, L. B., Paez-Espino, D., Staahl, B. T., Chen, J. S., Ma, E., Kyrpides, N. C., & Doudna, J. A. (2017) · Nature Communications · doi:10.1038/s41467-017-01408-4 · Cited by 205
CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming
Liu, Y., Chen, Y., Daley, T., Wang, F., Cao, W., Bhate, S. S., Lin, X., Still, C., Liu, H., Zhao, D., et al. (2018) · Cell stem cell · doi:10.1016/j.stem.2018.09.003 · Cited by 208
CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems
Jiang, W., & Marraffini, L. A. (2015) · Annual Review of Microbiology · doi:10.1146/annurev-micro-091014-104441 · Cited by 219
Kantor, B., Tagliafierro, L., Gu, J. T., Zamora, M. E., Ilich, E., Grenier, C., Huang, Z. Y., Murphy, S. K., & Chiba‐Falek, O. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.08.019 · Cited by 209
Rapidly evolving homing CRISPR barcodes
Kalhor, R., Mali, P., & Church, G. M. (2016) · Nature Methods · doi:10.1038/nmeth.4108 · Cited by 203
Synthetic Biology—The Synthesis of Biology
Ausländer, S., Ausländer, D., & Fussenegger, M. (2016) · Angewandte Chemie International Edition · doi:10.1002/anie.201609229 · Cited by 210
Challenges of CRISPR/Cas9 applications for long non-coding RNA genes
Goyal, A., Myacheva, K., Groß, M., Klingenberg, M., Arqué, B. D., & Diederichs, S. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw883 · Cited by 209
“CRISPRa / CRISPRi with dCas9-based transcription factors have been used for genome-wide gain- as well as lossof-function screens for protein-coding genes (58,59).”
MethodYe, L., Park, J. J.-Y., Peng, L., Yang, Q., Chow, R. D.-W., Dong, M. B., Lam, S. Z., Guo, J., Tang, E., Zhang, Y., et al. (2022) · Cell Metabolism · doi:10.1016/j.cmet.2022.02.009 · Cited by 208
“Although several studies demonstrated genome-scale GOF screen systems for protein-coding genes (Gilbert et al., 2014; Konermann et al., 2015) and long noncoding RNAs (lncRNAs) (Joung et al.”
BackgroundStructural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9
Anders, C., Bargsten, K., & Jinek, M. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.02.020 · Cited by 201
“Catalytically inactive variants of Cas9 have also been repurposed for RNAguided gene expression control and for cellular imaging applications (Chen et al., 2013; Gilbert et al., 2013; Konermann et al., 2015; Mali et al., 2013a).”
BackgroundCRISPR–Act3.0 for highly efficient multiplexed gene activation in plants
Pan, C., Wu, X., Markel, K., Malzahn, A. A., Kundagrami, N., Sretenovic, S., Zhang, Y., Cheng, Y., Shih, P. M., & Qi, Y. (2021) · Nature Plants · doi:10.1038/s41477-021-00953-7 · Cited by 206
CRISPR / dC as9 platforms in plants: strategies and applications beyond genome editing
Moradpour, M., & Abdulah, S. N. A. (2019) · Plant Biotechnology Journal · doi:10.1111/pbi.13232 · Cited by 205
“The SAM sgRNA enables the recruitment of a chimeric TAD consisting of the MS2 phage coat protein (MCP) (Konermann et al., 2015).”
BackgroundInfluentialDevelopment of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis
Westbrook, A. W., Moo‐Young, M., & Chou, C.-H. P. (2016) · Applied and Environmental Microbiology · doi:10.1128/aem.01159-16 · Cited by 205
“The CRISPRdCas9 system has been applied to genome-scale transcriptional repression (35) and activation (35, 36) for interrogation of gene function in human cell lines as well as to genetic and metabolic engineering of Escherichia coli (37, 38), Corynebacterium glutamicum (39), and mycobacteria (40).”
MethodSequence features associated with the cleavage efficiency of CRISPR/Cas9 system
Liu, X., Homma, A., Sayadi, J., Yang, S., Ohashi, J., & Takumi, T. (2016) · Scientific Reports · doi:10.1038/srep19675 · Cited by 166
Kung-Chun Chiu, D., Pui-Wah Tse, A., Law, C.-T., Ming-Jing Xu, I., Lee, D., Chen, M., Kit-Ho Lai, R., Wai-Hin Yuen, V., Wing-Sum Cheu, J., Wai-Hung Ho, D., et al. (2019) · Cell Death & Disease · doi:10.1038/s41419-019-2155-3 · Cited by 192
CRISPR-Cas9: a new and promising player in gene therapy
Xiao-Jie, L., Hui-Ying, X., Zun-Ping, K., Jin-Lian, C., & Li-Juan, J. (2015) · Journal of Medical Genetics · doi:10.1136/jmedgenet-2014-102968 · Cited by 200
Cytokines in cancer drug resistance: Cues to new therapeutic strategies
Jones, V. S., Huang, R.-Y., Chen, L.-P., Chen, Z., Fu, L., & Huang, R.-P. (2016) · Biochimica et Biophysica Acta (BBA) - Reviews on Cancer · doi:10.1016/j.bbcan.2016.03.005 · Cited by 199
Smith, J., Suresh, S., Schlecht, U., Wu, M., Wagih, O., Peltz, G., Davis, R. W., Steinmetz, L. M., Parts, L., & St.Onge, R. P. (2016) · Genome biology · doi:10.1186/s13059-016-0900-9 · Cited by 199
“wide libraries for knocking out [11–14], silencing [15], and activating genes [15, 16].”
Zhou, W., Cui, H., Ying, L., & Yu, X. (2018) · Angewandte Chemie International Edition · doi:10.1002/anie.201806941 · Cited by 194
Gene editing and CRISPR in the clinic: current and future perspectives
Hirakawa, M. P., Krishnakumar, R., Timlin, J. A., Carney, J. P., & Butler, K. S. (2020) · Bioscience Reports · doi:10.1042/bsr20200127 · Cited by 207
“adding multiple, distinct activation mechanisms (259) (Figure 6F).”
BackgroundCRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids
Hendriks, D. F. G., Clevers, H. C., & Artegiani, B. (2020) · Cell stem cell · doi:10.1016/j.stem.2020.10.014 · Cited by 202
Development of Light‐Activated CRISPR Using Guide RNAs with Photocleavable Protectors
Jain, P., Ramanan, V., Schepers, A. G., Dalvie, N. S., Panda, A., Fleming, H. E., & Bhatia, S. N. (2016) · Angewandte Chemie International Edition · doi:10.1002/anie.201606123 · Cited by 190
A CRISPR toolbox to study virus–host interactions
Puschnik, A. S., Majzoub, K., Ooi, Y. S., & Carette, J. E. (2017) · Nature Reviews Microbiology · doi:10.1038/nrmicro.2017.29 · Cited by 185
High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli
Cho, S., Choe, D., Lee, E., Kim, S. C., Palsson, B. Ø., & Cho, B. (2018) · ACS Synthetic Biology · doi:10.1021/acssynbio.7b00462 · Cited by 195
Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice
Krawczyk, K., Xue, S., Buchmann, P., Hamri, G. C., Saxena, P., Hussherr, M., Shao, J., Ye, H., Xie, M., & Fussenegger, M. (2020) · Science · doi:10.1126/science.aau7187 · Cited by 196
Platforms for Investigating LncRNA Functions
Richard, J. L. C., & Eichhorn, P. J. A. (2018) · SLAS TECHNOLOGY · doi:10.1177/2472630318780639 · Cited by 189
“Dimers of these MS2 coat proteins are recruited to MS2 RNA hairpins.(37) A recent genome-wide approach using a dual protein coding and noncoding integrated CRISPRa screen (DICaS) to identify functional coding and long noncoding RNA using the CaLR library (CRISPR activation of long noncoding RNA) revealed putative resi…”
BackgroundLong Noncoding RNA in Hematopoiesis and Immunity
Satpathy, A. T., & Chang, H. Y. (2015) · Immunity · doi:10.1016/j.immuni.2015.05.004 · Cited by 185
Metadata
- Authors
- Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., Hsu, P. D., Habib, N., Gootenberg, J. S., Nishimasu, H., Nureki, O., & Zhang, F.
- Affiliations
- Broad Institute (US)
- The University of Tokyo (JP)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature
- Published
- 2014-12-10
- Volume
- 517
- Issue
- 7536
- Pages
- 583-588
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4420636
- OpenAlex
- W2127862779
- Cited by
- 2,825
- References
- 52
- Citations by year
- 2026: 154 · 2025: 204 · 2024: 266 · 2023: 236 · 2022: 279 · 2021: 281 · 2020: 301 · 2019: 270 · 2018: 261 · 2017: 229 · 2016: 214 · 2015: 122 · 2014: 3
- FWCI
- 77.58
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- PMC
- Funders
- Japan Society for the Promotion of Science (26291010)
- National Institute of Mental Health (DP1MH100706)
- National Institute of Neurological Disorders and Stroke (R01NS062849, R01NS073124)
- National Center for Chronic Disease Prevention and Health Promotion (DP1‑MH100706)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, transcriptional activation, long intergenic non-coding RNA, gain-of-function screen, BRAF inhibitor resistance, melanoma, drug resistance, gene expression signatures, gene perturbation
- MeSH terms
- Genetic Engineering, Genetic Testing, Humans, Indoles, Melanoma, Sulfonamides, Reproducibility of Results, Transcriptional Activation, Gene Library, Up-Regulation, Genome, Human, Gene Expression Regulation, Neoplastic, DNA, Complementary, Drug Resistance, Neoplasm, RNA, Untranslated, Cell Line, Tumor, Proto-Oncogene Proteins B-raf, Genetic Loci, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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