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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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“Other screening technologies, such as Cas9-mediated activation (Gilbert et al., 2014; Konermann et al., 2015), can identify metastasis-regulating factors that act in a gain-of-function manner.”
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“Furthermore, targeting the lncRNA Firre by CRISPR/Cas9 delays Ras-induced cellular senescence.(192,193) Evidence shows that circRNAs play important roles in the modulation of aging and aging-related diseases, such as cardiovascular disorders, diabetes, and neurodegenerative diseases.”
BackgroundDiverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems
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“Other applications of Cas9 include building gene circuits [141-143], creating new anti-microbials [144], antivirals [145-147], and large-scale gain- and loss-of-function screening [148-151] (Table 1).”
BackgroundSelf-citationFunctional annotation of native enhancers with a Cas9–histone demethylase fusion
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A Multifunctional AAV-CRISPR-Cas9 and Its Host Response
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“CRISPR/Cas9 knockout screens are better than previous functional shRNA knockdown screens (Berns et al. 2004; Luo et al. 2008) for the study of phenotypes that require complete inactivation of genes (Shalem et al. 2014).”
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“Finally, the widely used synergistic activation mediator (SAM) system recruits the two transcription factors HSF1 and p65 to enhance gene activation on top of a dCas9–VP64 fusion 61 .”
MethodDirect cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing
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Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting
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“In addition to knock-out screens, proof-of-concept CRISPR-activator or inhibitor screens using dCas9 and genome-scale sgRNA libraries have also been successfully conducted (30, 31).”
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“…have been successfully derived by fusions with functional protein that bind specific loci, or to activate or repress gene activity at the target site (Chen et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Mandegar et al., 2016; Tanenbaum et al., 2014) (CRISPRa and CRISPRi, Figure 2).”
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“Alter-natively, activation of gene expression through open reading frame (ORF) or in vitro and in vivo CRISPR activation (CRISPRa) screens has enabled the discovery of new and distinct synthetic lethal interactions that are not detected in knockout screening approaches 225,227–231 .”
Comparison of Cas9 activators in multiple species
Chavez, A., Tuttle, M. L., Pruitt, B. W., Ewen‐Campen, B., Chari, R., Ter‐Ovanesyan, D., Haque, S. J., Cecchi, R. J., Kowal, E. J. K., Buchthal, J., et al. (2016) · Nature Methods · doi:10.1038/nmeth.3871 · Cited by 521
Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR
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Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR
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“For example in melanoma activation dataset [5], cell lines underwent different sgRNA expression selection methods (cells are first selected using puromycin or zeocin), duration of treatment (3-day or 21-day treatment) and drug treatments (DMSO or PLX).”
BackgroundMethodInfluentialAn enhanced CRISPR repressor for targeted mammalian gene regulation
Yeo, N. C., Chavez, A., Lance‐Byrne, A., Chan, Y., Menn, D. J., Milanova, D., Kuo, C., Guo, X., Sharma, S., Tung, A., et al. (2018) · Nature Methods · doi:10.1038/s41592-018-0048-5 · Cited by 489
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Synthetic biology to access and expand nature's chemical diversity
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Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells
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Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow
Ma, H., Tu, L., Naseri, A., Huisman, M., Zhang, S., Grünwald, D., & Pederson, T. (2016) · Nature Biotechnology · doi:10.1038/nbt.3526 · Cited by 472
Am I ready for CRISPR? A user's guide to genetic screens
Doench, J. G. (2018) · Nature Reviews Genetics · doi:10.1038/nrg.2017.97 · Cited by 452
A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks
Parnas, O., Jovanovic, M., Eisenhaure, T. M., Herbst, R. H., Dixit, A., Ye, C. J., Przybylski, D., Platt, R. J., Tirosh, I., Sanjana, N. E., et al. (2015) · Cell · doi:10.1016/j.cell.2015.06.059 · Cited by 440
“…short palindromic repeats (CRISPR)-associated nuclease Cas9 system has enabled pooled genome-wide screens of gene function (Gilbert et al., 2014; Konermann et al., 2015; Shalem et al., 2014; Wang et al., Cell 162, 675–686, July 30, 2015 ª2015 Elsevier Inc. 675 (A) Flow cytometry of…”
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Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
Replogle, J. M., Norman, T. M., Xu, A., Hussmann, J. A., Chen, J., Cogan, J. Z., Meer, E. J., Terry, J. M., Riordan, D. P., Srinivas, N., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0470-y · Cited by 465
Role of non-coding sequence variants in cancer
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In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation
Liao, H.-K., Hatanaka, F., Araoka, T., Reddy, P., Wu, M.-Z., Sui, Y., Yamauchi, T., Sakurai, M., O’Keefe, D. D., Núñez-Delicado, E., et al. (2017) · Cell · doi:10.1016/j.cell.2017.10.025 · Cited by 451
“To this end, we adapted the SAMmodule from second-generation CRISPR/Cas9-based TGA systems (Konermann et al., 2015).”
InfluentialPromoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element
Cho, S. W., Xu, J., Sun, R., Mumbach, M. R., Carter, A. C., Chen, Y. G., Yost, K. E., Kim, J., He, J., Nevins, S. A., et al. (2018) · Cell · doi:10.1016/j.cell.2018.03.068 · Cited by 459
Massively parallel Cas13 screens reveal principles for guide RNA design
Wessels, H.-H., Méndez-Mancilla, A., Guo, X., Legut, M., Daniloski, Z., & Sanjana, N. E. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0456-9 · Cited by 418
Editing the epigenome: technologies for programmable transcription and epigenetic modulation
Thakore, P. I., Black, J. B., Hilton, I. B., & Gersbach, C. A. (2016) · Nature Methods · doi:10.1038/nmeth.3733 · Cited by 451
Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute
Wang, B., Wang, M., Zhang, W., Xiao, T., Chen, C.-H., Wu, A., Wu, F., Traugh, N. C., Wang, X., Li, Z., et al. (2019) · Nature Protocols · doi:10.1038/s41596-018-0113-7 · Cited by 444
CRISPR technologies for precise epigenome editing
Nakamura, M., Gao, Y., Dominguez, A. A., & Qi, L. S. (2021) · Nature Cell Biology · doi:10.1038/s41556-020-00620-7 · Cited by 456
Cornerstones of CRISPR–Cas in drug discovery and therapy
Fellmann, C., Gowen, B. G., Lin, P., Doudna, J. A., & Corn, J. E. (2016) · Nature Reviews Drug Discovery · doi:10.1038/nrd.2016.238 · Cited by 452
Direct cell reprogramming: approaches, mechanisms and progress
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Engineering the next generation of cell-based therapeutics
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Crystal Structure of Staphylococcus aureus Cas9
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“In the SpCas9 structure, the tetraloop and stem loop 2 of the sgRNA are exposed to the solvent (Nishimasu et al., 2014; Anders et al., 2014) (Figure S4D), and permitted the insertion of RNA aptamers into the sgRNA to create robust RNAguided transcription activators (Konermann et al., 2015).”
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Katti, A., Diaz, B. J., Caragine, C. M., Sanjana, N. E., & Dow, L. E. (2022) · Nature reviews. Cancer · doi:10.1038/s41568-022-00441-w · Cited by 457
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Self-citationGenome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood
Joung, J., Engreitz, J., Konermann, S., Abudayyeh, O. O., Verdine, V. K., Aguet, F., Gootenberg, J. S., Sanjana, N. E., Wright, J. B., Fulco, C. P., et al. (2017) · Nature · doi:10.1038/nature23451 · Cited by 427
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“Importantly, because of the ease with which the CRISPRCas9 system can be used, genome-wide screens using Cas9 transcriptional activators (Gilbert et al. 2014; Konermann et al. 2015) and repressors (Gilbert et al.”
BackgroundEngineered miniature CRISPR-Cas system for mammalian genome regulation and editing
Xu, X., Chemparathy, A. G., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., & Qi, L. S. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.08.008 · Cited by 411
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities
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A Single-Cell Transcriptomic Atlas of Human Skin Aging
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Divergent lncRNAs Regulate Gene Expression and Lineage Differentiation in Pluripotent Cells
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Expanding the Biologist’s Toolkit with CRISPR-Cas9
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“…proteins through engineered molecular scaffolds fused to Cas9 (Gilbert et al., 2014; Tanenbaum et al., 2014) or RNA aptamers fused to sgRNA (Konermann et al., 2015; Mali et al., 2013a). dCas9 can also be used to probe and manipulate the genome in other ways that ultimately rely on specific…”
BackgroundApplications of the CRISPR-Cas9 system in cancer biology
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Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems
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The emerging and uncultivated potential of CRISPR technology in plant science
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Exploring genetic interaction manifolds constructed from rich single-cell phenotypes
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Manghwar, H., Li, B., Ding, X., Hussain, A., Lindsey, K., Zhang, X., & Jin, S. (2020) · Advanced Science · doi:10.1002/advs.201902312 · Cited by 347
A CRISPR-based approach for targeted DNA demethylation
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CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine
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Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells
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“…#12259 psPAX2 from Didier Trono Addgene plasmid #12260 lenti-dCas9-VP64-Blast Konermann et al., 2015 Addgene plasmid #61425 lenti-sgRNA(MS2)-puro Konermann et al., 2015 Addgene plasmid #73795 lenti-dCas9-KRAB-Blast This paper Addgene plasmid #89567 Lenti-sgRNA(MS2)-puro-barcode This paper…”
BackgroundMethodOptimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
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“These results also suggest that the optimized sgRNAs would perform better for CRISPR-Cas9-based genome-wide pooled screenings, which use lentivirus to deliver sgRNAs at low MOI [13–20].”
BackgroundDeregulation of ribosomal protein expression and translation promotes breast cancer metastasis
Ebright, R. Y., Lee, S., Wittner, B. S., Niederhoffer, K., Nicholson, B. T., Bardia, A., Truesdell, S. S., Wiley, D. F., Wesley, B., Li, S. S., et al. (2020) · Science · doi:10.1126/science.aay0939 · Cited by 378
A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
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“The authors speculate that perhaps the dissociation of genomic DNA caused by the RNADNA interactions mediated by the guide RNA affected nearby transcription complexes; they suggest that new dCas9-based activator platforms may show more robust transcriptional activity [105].”
BackgroundCRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter
Choudhury, S. R., Cui, Y., Lubecka, K., Stefañska, B., & Irudayaraj, J. (2016) · Oncotarget · doi:10.18632/oncotarget.10234 · Cited by 355
“In particular, catalytically inactive Cas9 (dCas9) in combination with single-guide RNA (sgRNA) are frequently being fused to various transcriptional factors (TFs) such as KRAB-repressors or VP64 activators to respectively inactivate (CRISPRi) or activate (CRISPRa) expression of a given gene [22, 23].”
Background
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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