Highly efficient Cas9-mediated transcriptional programming
Open accessPublished version of a preprint
Abstract
The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
CRISPRi 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 [29]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Multiplex 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 · see citing works
Self-citationRepurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016 · see citing works
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., et al. (2013) · Cell · doi:10.1016/j.cell.2013.06.044 · Cited by 3,696 · see citing works
Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
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., et al. (2015) · Nature · doi:10.1038/nature14136 · Cited by 2,825 · see citing works
RNA-programmed genome editing in human cells
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013) · eLife · doi:10.7554/elife.00471 · Cited by 2,098 · see citing works
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · see citing works
Cas9 as a versatile tool for engineering biology
Mali, P., Esvelt, K. M., & Church, G. M. (2013) · Nature Methods · doi:10.1038/nmeth.2649 · Cited by 1,197 · see citing works
Self-citationA public genome-scale lentiviral expression library of human ORFs
Yang, X., Boehm, J. S., Yang, X., Salehi-Ashtiani, K., Hao, T., Shen, Y., Lubonja, R., Thomas, S. R., Alkan, O., Bhimdi, T., et al. (2011) · Nature Methods · doi:10.1038/nmeth.1638 · Cited by 570 · see citing works
The Mediator complex and transcription regulation
Poss, Z. C., Ebmeier, C. C., & Taatjes, D. J. (2013) · Critical Reviews in Biochemistry and Molecular Biology · doi:10.3109/10409238.2013.840259 · Cited by 364 · see citing works
Flagged first, then by citations
Research works printed but not linked: 15
- [1]Garneau JE The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Nature 468 67 71 2010 21048762
- [3]Mojica FJM Díez-Villaseñor C García-Martínez J Almendros C Short motif sequences determine the targets of the prokaryotic CRISPR defence system Microbiol Read Engl 155 733 740 2009 19246744
- [5]Mali P RNA-guided human genome engineering via Cas9 Science 339 823 826 2013 23287722 PMC3712628
- [7]Sapranauskas R The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli Nucleic Acids Res 39 9275 9282 2011 21813460 PMC3241640
- [9]Maeder ML CRISPR RNA-guided activation of endogenous human genes Nat Methods 10 977 979 2013 23892898 PMC3794058
- [10]Perez-Pinera P RNA-guided gene activation by CRISPR-Cas9-based transcription factors Nat Methods 10 973 976 2013 23892895 PMC3911785
- [12]Hsu PD Lander ES Zhang F Development and Applications of CRISPR-Cas9 for Genome Engineering Cell 157 1262 1278 2014 24906146 PMC4343198
- [13]Gersbach CA Genome engineering: the next genomic revolution Nat Methods 11 1009 1011 2014 25264777
- [15]Esvelt KM Orthogonal Cas9 proteins for RNA-guided gene regulation and editing Nat Methods 10 1116 1121 2013 24076762 PMC3844869
- [17]Busskamp V Rapid neurogenesis through transcriptional activation in human stem cells Mol Syst Biol 10 760 2014 25403753 PMC4299601
- [18]Zhang Y Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells Neuron 78 785 798 2013 23764284 PMC3751803
- [19]Tanenbaum ME Gilbert LA Qi LS Weissman JS Vale RD A protein-tagging system for signal amplification in gene expression and fluorescence imaging Cell 159 635 646 2014 25307933 PMC4252608
- [20]Zalatan JG Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds Cell 160 339 350 2015 25533786 PMC4297522
- [21]Alberti S Gitler AD Lindquist S A suite of Gateway® cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae Yeast 24 913 919 2007 17583893 PMC2190539
- [22]DiCarlo JE Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems Nucleic Acids Res 41 4336 4343 2013 23460208 PMC3627607
Cited by
Sorted by citations
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
Hu, J. H., Miller, S. M., Geurts, M. H., Tang, W., Chen, L., Sun, N., Zeina, C. M., Gao, X., Rees, H. A., Lin, Z., et al. (2018) · Nature · doi:10.1038/nature26155 · Cited by 1,552 · see citing works
Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
Joung, J., Konermann, S., Gootenberg, J. S., Abudayyeh, O. O., Platt, R. J., Brigham, M. D., Sanjana, N. E., & Zhang, F. (2017) · Nature Protocols · doi:10.1038/nprot.2017.016 · Cited by 1,243 · see citing works
Thakore, P. I., D'Ippolito, A. M., Song, L., Safi, A., Shivakumar, N. K., Kabadi, A. M., Reddy, T. E., Crawford, G. E., & Gersbach, C. A. (2015) · Nature Methods · doi:10.1038/nmeth.3630 · Cited by 1,012 · see citing works
Horlbeck, M. A., Gilbert, L. A., Villalta, J. E., Adamson, B., Pak, R. A., Chen, Y., Fields, A. P., Park, C. Y., Corn, J. E., Kampmann, M., et al. (2016) · eLife · doi:10.7554/elife.19760 · Cited by 894 · see citing works
Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation
Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2015.2 · Cited by 832 · see citing works
Engineered pegRNAs improve prime editing efficiency
Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A., Chen, P. J., Anzalone, A. V., An, M., Newby, G. A., Chen, J. C., Hsu, A., et al. (2022) · Nature Biotechnology · doi:10.1038/s41587-021-01039-7 · Cited by 727 · see citing works
Genome-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
A Multifunctional AAV-CRISPR-Cas9 and Its Host Response
Chew, W. L., Tabebordbar, M., Cheng, J. K. W., Mali, P., Wu, E. Y., Ng, A. H. M., Zhu, K., Wagers, A. J., & Church, G. M. (2016) · Nature Methods · doi:10.1038/nmeth.3993 · Cited by 617 · see citing works
Self-citationIn 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 · see citing works
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457 · see citing works
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 · see citing works
Mangeot, P. E., Risson, V., Fusil, F., Marnef, A., Laurent, E., Blin, J., Mournetas, V., Massouridès, E., Sohier, T. J. M., Corbin, A., et al. (2019) · Nature Communications · doi:10.1038/s41467-018-07845-z · Cited by 346 · see citing works
CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency
Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., Hardin, A., Eckalbar, W. L., Vaisse, C., & Ahituv, N. (2019) · Science · doi:10.1126/science.aau0629 · Cited by 323 · see citing works
Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
Lian, J., HamediRad, M., Hu, S., & Zhao, H. (2017) · Nature Communications · doi:10.1038/s41467-017-01695-x · Cited by 319 · see citing works
Gene therapy for neurological disorders: progress and prospects
Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M., & Sah, D. W. Y. (2018) · Nature Reviews Drug Discovery · doi:10.1038/nrd.2018.110 · Cited by 306 · see citing works
Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens
Najm, F. J., Strand, C., Donovan, K. F., Hegde, M., Sanson, K. R., Vaimberg, E. W., Sullender, M. E., Hartenian, E., Kalani, Z., Fusi, N., et al. (2018) · Nature Biotechnology · doi:10.1038/nbt.4048 · Cited by 279 · see citing works
Zhou, H., Liu, J., Zhou, C., Gao, N., Rao, Z., Li, H., Hu, X., Li, C., Yao, X., Shen, X., et al. (2018) · Nature Neuroscience · doi:10.1038/s41593-017-0060-6 · Cited by 276 · see citing works
Freed-Pastor, W. A., Lambert, L. J., Ely, Z. A., Pattada, N. B., Bhutkar, A., Eng, G., Mercer, K. L., Garcia, A. P., Lin, L., Rideout, W. M., et al. (2021) · Cancer Cell · doi:10.1016/j.ccell.2021.07.007 · Cited by 273 · see citing works
Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges
Taha, E. A., Lee, J., & Hotta, A. (2022) · Journal of Controlled Release · doi:10.1016/j.jconrel.2022.01.013 · Cited by 259 · see citing works
Robust 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 · see citing works
Human pluripotent reprogramming with CRISPR activators
Weltner, J., Balboa, D., Katayama, S., Bespalov, M., Krjutškov, K., Jouhilahti, E.-M., Trokovic, R., Kere, J., & Otonkoski, T. (2018) · Nature Communications · doi:10.1038/s41467-018-05067-x · Cited by 181 · see citing works
Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
Roth, T. L., Li, P. J., Blaeschke, F., Nies, J. F., Apathy, R., Mowery, C., Yu, R., Nguyen, M. L. T., Lee, Y., Truong, A., et al. (2020) · Cell · doi:10.1016/j.cell.2020.03.039 · Cited by 186 · see citing works
A dopamine-induced gene expression signature regulates neuronal function and cocaine response
Savell, K. E., Tuscher, J. J., Zipperly, M. E., Duke, C. G., Phillips, R. A., Bauman, A. J., Thukral, S., Sultan, F. A., Goska, N. A., Ianov, L., et al. (2020) · Science Advances · doi:10.1126/sciadv.aba4221 · Cited by 180 · see citing works
CRISPR-based functional genomics for neurological disease
Kampmann, M. (2020) · Nature Reviews Neurology · doi:10.1038/s41582-020-0373-z · Cited by 163 · see citing works
In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation
Moreno, A. M., Fu, X., Zhu, J., Katrekar, D., Shih, Y.-R. V., Marlett, J., Cabotaje, J., Tat, J., Naughton, J., Lisowski, L., et al. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2018.04.017 · Cited by 159 · see citing works
Current status and perspectives of genome editing technology for microalgae
Jeon, S., Lim, J.-M., Lee, H.-G., Shin, S.-E., Kang, N. K., Park, Y.-I., Oh, H.-M., Jeong, W.-J., Jeong, B.-R., & Chang, Y. K. (2017) · Biotechnology for Biofuels · doi:10.1186/s13068-017-0957-z · Cited by 154 · see citing works
Single-Nucleotide-Resolution Computing and Memory in Living Cells
Farzadfard, F., Gharaei, N., Higashikuni, Y., Jung, G., Cao, J., & Lu, T. K. (2019) · Molecular Cell · doi:10.1016/j.molcel.2019.07.011 · Cited by 122 · see citing works
Transcriptional 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 · see citing works
Recent advances in gene therapy for neurodevelopmental disorders with epilepsy
Turner, T. J., Zourray, C., Schorge, S., & Lignani, G. (2021) · Journal of Neurochemistry · doi:10.1111/jnc.15168 · Cited by 71 · see citing works
Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies
McTague, A., Rossignoli, G., Ferrini, A., Barral, S., & Kurian, M. A. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.630600 · Cited by 67 · see citing works
Tycko, J., Van, M. V., Aradhana, DelRosso, N., Ye, H., Yao, D., Valbuena, R., Vaughan-Jackson, A., Xu, X., Ludwig, C., et al. (2025) · Nature Biotechnology · doi:10.1038/s41587-024-02442-6 · Cited by 57 · see citing works
Rational design of a compact CRISPR-Cas9 activator for AAV-mediated delivery
Vora, S., Cheng, J., Xiao, R., VanDusen, N. J., Quintino, L., Pu, W. T., Vandenberghe, L. H., Chavez, A., & Church, G. (2018) · doi:10.1101/298620 · Cited by 42 · see citing works
Self-citationLo, Y.-H., Horn, H. T., Huang, M.-F., Yu, W.-C., Young, C.-M., Liu, Q., Tomaske, M., Towers, M., Dominguez, A., Bassik, M. C., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-62818-3 · Cited by 37 · see citing works
Lohia, A., Sahel, D. K., Salman, M., Singh, V., Mariappan, I., Mittal, A., & Chitkara, D. (2022) · Asian Journal of Pharmaceutical Sciences · doi:10.1016/j.ajps.2022.02.001 · Cited by 33 · see citing works
Hamilton, P. J., Lim, C. J., Nestler, E. J., & Heller, E. A. (2018) · Methods in Molecular Biology · doi:10.1007/978-1-4939-7774-1_5 · Cited by 28 · see citing works
- Authors
- Chavez, A., Scheiman, J., Vora, S., Pruitt, B. W., Tuttle, M., P R Iyer, E., Lin, S., Kiani, S., Guzman, C. D., Wiegand, D. J., Ter-Ovanesyan, D., Braff, J. L., Davidsohn, N., Housden, B. E., Perrimon, N., Weiss, R., Aach, J., Collins, J. J., & Church, G. M.
- Affiliations
- Harvard University (US)
- Howard Hughes Medical Institute (US)
- Massachusetts Institute of Technology (US)
- Broad Institute (US)
- Corresponding author
- Church, George M
- ORCID
- Venue
- Nature Methods
- Published
- 2015-03-02
- Volume
- 12
- Issue
- 4
- Pages
- 326-328
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4393883
- OpenAlex
- W2163173465
- Cited by
- 1,622
- References
- 10
- Citations by year
- 2026: 114 · 2025: 152 · 2024: 169 · 2023: 158 · 2022: 182 · 2021: 190 · 2020: 158 · 2019: 150 · 2018: 139 · 2017: 101 · 2016: 81 · 2015: 24 · 2014: 1
- FWCI
- 49.96
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- PMC
- Funders
- National Science Foundation (DE-FG02-02ER63445)
- U.S. Department of Energy (DE‐FG02‐, DEFG0202ER63445, FG02-02ER63445)
- Massachusetts Institute of Technology
- National Institutes of Health (DE-FG02-02ER63445, 5t32gm007598-41)
- Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
- National Human Genome Research Institute (P50HG005550, HG005550)
- National Cancer Institute (R01CA173712, T32CA009216)
- National Institute of General Medical Sciences (T32-GM007598)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- Cas9, transcriptional activation, transcription factors, gene activation, human induced pluripotent stem cells, neuronal differentiation, RNA-guided nucleases
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cell Differentiation, Endonucleases, Genetic Techniques, Humans, Neurons, Staphylococcus aureus, Transcriptional Activation, RNA, Guide, Kinetoplastida, Induced Pluripotent Stem Cells, HEK293 Cells
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
