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.
“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]
The Extended Data Figure 7 caption describes three lentiviral vectors expressing sgRNA, dCas9-VP64, and MS2-p65-HSF1, not a dual rAAV system expressing dCas9-VPR and gRNA. The genome-scale gene activation screen section likewise identifies the delivery system as lentiviral. These passages do not give a 4277-bp dCas9-VPR cDNA size. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredCRISPRi and CRISPRa technologies utilize catalytically inactive Cas9 (dCas9) fused to either a transcriptional repressor (e.g., KRAB) or a transcriptional activator (e.g., VPR).
“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]
The Abstract describes catalytically inactive Cas9 fused to transcriptional activation domains, and the Structure-guided design section identifies the paper’s activator as dCas9-VP64 in the SAM system. The cited paper therefore supports the CRISPRa portion, but our copy does not describe the VPR fusion or a dCas9-KRAB CRISPRi system. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe dCas9-effector strategy has been applied in a genome-wide screening study in which gain-of-function mutations in melanoma cells that confer drug resistance were identified.
“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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“…while several strategies have been described for CRISPR-mediated activation (Chavez et al., 2015; Gilbert et al., 2013; Hilton et al., 2015; Konermann et al., 2015; Maeder et al., 2013; PerezPinera et al., 2013; Tanenbaum et al., 2014; Zalatan et al., 2015), a recent comparison of these…”
BackgroundInfluentialCRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes
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Applications of CRISPR technologies in research and beyond
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Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis
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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.”
BackgroundSelf-citationBeyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation
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Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA
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“Following the initial demonstration of the feasibility of using Cas9 to edit mammalian genomes, there was a burst of efforts to further adapt this endonuclease for a range of applications, from high-throughput gain-of-function screening (Gilbert et al., 2014; Konermann et al., 2015) to targeted modulation of histone m…”
BackgroundInfluentialSelf-citationGenome-wide programmable transcriptional memory by CRISPR-based epigenome editing
Nuñez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., et al. (2021) · Cell · doi:10.1016/j.cell.2021.03.025 · Cited by 760 · see citing works
“When cells reached 60%–80% confluency the next day, cells were transfected with 500 ng of dCas9 plasmid (dCas9 or TETv1-4) and 300 ng of sgRNA-transactivator plasmid (sgRNA only, VP64, p65, Rta, VP64-p65, p65-Rta, or VPR) using TransIT-LT1 Transfection Reagent (Mirus) and Opti-MEMTM I Reduced SerumMedium (Thermo Fishe…”
BackgroundMethodInfluentialFerroptosis surveillance independent of GPX4 and differentially regulated by sex hormones
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CRISPR/Cas9 therapeutics: progress and prospects
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A split-Cas9 architecture for inducible genome editing and transcription modulation
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“In addition, strategies employing catalytically inactive Cas9 can direct effector proteins to genomic targets 3–5 to modulate transcription.”
BackgroundSelf-citationA CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
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“SSNs can induce sequence-specific DNA double-strand breaks (DSBs), which are subsequently repaired by either nonhomologous end joining (NHEJ) or homologous recombination (Kanaar et al., 1998; Puchta, 2005).”
BackgroundEpigenetic regulation of aging: implications for interventions of aging and diseases
Kang, W., Liu, H., Hu, Q., Wang, L., Liu, J., Zheng, Z., Zhang, W., Ren, J., Zhu, F., & Liu, G. (2022) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-022-01211-8 · Cited by 694 · see citing works
“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
Mohanraju, P., Makarova, K. S., Zetsche, B., Zhang, F., Koonin, E. V., & John van der, O. O. S. T. (2016) · Science · doi:10.1126/science.aad5147 · Cited by 668 · see citing works
“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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Synthetic lethality and cancer
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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).”
BackgroundSelf-citationExosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs
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Chemoresistance in Pancreatic Cancer
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Self-citationPast, present, and future of CRISPR genome editing technologies
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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).”
The CRISPR-Cas immune system: Biology, mechanisms and applications
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CRISPR/Cas system: recent advances and future prospects for genome editing
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Induced Pluripotent Stem Cells Meet Genome Editing
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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).”
BackgroundThe present and future of the Cancer Dependency Map
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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
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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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BackgroundMethodInfluentialAn enhanced CRISPR repressor for targeted mammalian gene regulation
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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
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Am I ready for CRISPR? A user's guide to genetic screens
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A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks
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“…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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Role of non-coding sequence variants in cancer
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“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
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Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute
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CRISPR technologies for precise epigenome editing
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Cornerstones of CRISPR–Cas in drug discovery and therapy
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Direct cell reprogramming: approaches, mechanisms and progress
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Self-citationEngineering 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).”
BackgroundSelf-citationCRISPR in cancer biology and therapy
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Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood
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Self-citationGenome-Editing Technologies: Principles and ApplicationsPreprint
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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 · see citing works
Exploring genetic interaction manifolds constructed from rich single-cell phenotypes
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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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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
Dang, Y., Jia, G., Choi, J., Ma, H., Anaya, E., Ye, C., Shankar, P., & Wu, H. (2015) · Genome biology · doi:10.1186/s13059-015-0846-3 · Cited by 364 · see citing works
“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 365 · see citing works
A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
Zhou, W., Li, H., Ying, L., Zhao, Z., Chu, P. K.-H., & Yu, X. (2018) · Nature Communications · doi:10.1038/s41467-018-07324-5 · Cited by 358 · see citing works
Tak, Y. G., & Farnham, P. (2015) · Epigenetics & Chromatin · doi:10.1186/s13072-015-0050-4 · Cited by 342 · see citing works
“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 352 · see citing works
“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
- 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 · 2013: 2
- 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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