Cited by 1,752CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

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Mali, Prashant, Aach, John, Stranges, P Benjamin, Esvelt, Kevin M, Moosburner, Mark, Kosuri, Sriram, Yang, Luhan, Church, George M·2013·Nature Biotechnology

DOI 10.1038/nbt.2675PMC PMC3818127

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Abstract

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

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Background
RNA-Guided Human Genome Engineering via Cas9
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BackgroundMethod
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Method
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Background
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Background
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BackgroundMethod
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Background
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Background
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Background
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Influential
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Background
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Background
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Background
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Editing the Mouse Genome Using the CRISPR–Cas9 SystemPreprint
Williams, A., Henao‐Mejia, J., & Flavell, R. A. (2016) · Cold Spring Harbor Protocols · doi:10.1101/pdb.top087536 · Cited by 22
“The repurposing of Cas9 to generate site-specific DSB in mammalian genomes was a turning point in genome editing (Cho et al. 2013; Cong et al. 2013; Mali et al. 2013b; Wang et al. 2013; Yang et al. 2013).”
Recent progress in prompt molecular detection of liquid biopsy using Cas enzymes: innovative approaches for cancer diagnosis and analysis
Yazdi, Z. F., Roshannezhad, S., Sharif, S., & Abbaszadegan, M. R. (2024) · Journal of Translational Medicine · doi:10.1186/s12967-024-05908-y · Cited by 22
Proven and novel strategies for efficient editing of the human genome
Mussolino, C., Mlambo, T., & Cathomen, T. (2015) · Current Opinion in Pharmacology · doi:10.1016/j.coph.2015.08.008 · Cited by 21
RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts
Xiong, K., Zhou, Y., Blichfeld, K. A., Hyttel, P., Bolund, L., Freude, K., & Luo, Y. (2017) · Cellular Reprogramming · doi:10.1089/cell.2017.0006 · Cited by 21
“…provided an open-resource and powerful tool for targeting human reprogramming factors including KLF4 , LIN28 , MYC , OCT4 , NANOG , and SOX2 in various human cell types (Cheng et al., 2013, 2016; Mali et al., 2013; Perez-Pinera et al., 2013; Hu et al., 2014; Ji et al., 2014; Balboa et al., 2015).”
Targeted genome editing in Caenorhabditis elegans using CRISPR /Cas9
Farboud, B. (2017) · Wiley Interdisciplinary Reviews Developmental Biology · doi:10.1002/wdev.287 · Cited by 20
Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening
Aregger, M., Xing, K., & Gonatopoulos-Pournatzis, T. (2021) · Nature Protocols · doi:10.1038/s41596-021-00595-1 · Cited by 22
A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila
Amo, V. L. D., Juste, S. S., & Gantz, V. M. (2022) · Cell Reports · doi:10.1016/j.celrep.2022.110843 · Cited by 21
“Nicks induced by nD10A promoted higher HDR rates than nH840A, in which HDR was almost undetectable (Bothmer et al., 2017; Hyodo et al., 2020; Mali et al., 2013; Wang et al., 2018, 2021).”
CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements
Nasrallah, A., Sulpice, E., Kobaisi, F., Gidrol, X., & Rachidi, W. (2022) · Cells · doi:10.3390/cells11223615 · Cited by 26
Principles and applications of TAL effectors for plant physiology and metabolism
Bogdanove, A. J. (2014) · Current Opinion in Plant Biology · doi:10.1016/j.pbi.2014.05.007 · Cited by 21
Fast and EfficientDrosophila melanogasterGene Knock-Ins Using MiMIC Transposons
Vilain, S., Vanhauwaert, R., Maes, I., Schoovaerts, N., Zhou, L., Soukup, S., da Cunha, R., Lauwers, E., Fiers, M. W. E. J., & Verstreken, P. (2014) · G3 Genes Genomes Genetics · doi:10.1534/g3.114.014803 · Cited by 21
Use of CRISPR/Cas9 to model brain diseases
Yan, S., Tu, Z., Li, S., & Li, X. (2017) · Progress in Neuro-Psychopharmacology and Biological Psychiatry · doi:10.1016/j.pnpbp.2017.04.003 · Cited by 20
“Research has shown that Cas9 can tolerate mismatches, depending on their distribution and number (Hsu et al., 2013; Mali et al., 2013; Fu et al., 2014).”
Genome Engineering Tools in Plant Synthetic Biology
Mohan, K., Ghose, K., Rai, A., Singh, H., Srivastava, R., & Mendu, V. (2019) · Elsevier eBooks · doi:10.1016/b978-0-444-64085-7.00003-4 · Cited by 21
Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing
Cai, W., & Wang, M. (2019) · 中国科学通报：英文版 · doi:10.1016/j.scib.2019.07.035 · Cited by 21
Genome Editing with CRISPR‐Cas: An Overview
Robb, G. B. (2019) · Current Protocols Essential Laboratory Techniques · doi:10.1002/cpet.36 · Cited by 24
Computational design of anti-CRISPR proteins with improved inhibition potency
Mathony, J., Harteveld, Z., Schmelas, C., zu Belzen, J. U., Aschenbrenner, S., Sun, W. Y., Hoffmann, M. D., Stengl, C., Scheck, A., Georgeon, S., et al. (2020) · Nature Chemical Biology · doi:10.1038/s41589-020-0518-9 · Cited by 21
Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation
Fu, Y., Yang, X., Li, S., Ma, C., An, Y., Cheng, T., Liang, Y., Sun, S., Cheng, T., Zhao, Y., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-56735-8 · Cited by 22
New horizons in genome engineering of Drosophila melanogaster
Kondo, S. (2014) · Genes & Genetic Systems · doi:10.1266/ggs.89.3 · Cited by 20
“Both in vivo and in vitro, several studies have shown that Cas9 is capable of cleaving a target site with up to five mismatches albeit with reduced efficiency (Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013).”
Background
Novel Functional Genomics Approaches: A Promising Future in the Combat Against Plant Viruses
Fondong, V. N., Nagalakshmi, U., & Dinesh‐Kumar, S. P. (2016) · Phytopathology · doi:10.1094/phyto-03-16-0145-fi · Cited by 20
CRISPR Technology for Ocular Angiogenesis
Chung, S. H., Sin, T.-N., Ngo, T., & Yiu, G. C. (2020) · Frontiers in Genome Editing · doi:10.3389/fgeed.2020.594984 · Cited by 21
Chromatin Alterations in Neurological Disorders and Strategies of (Epi)Genome Rescue
Janowski, M., Milewska, M., Zare, P., & Pękowska, A. (2021) · Pharmaceuticals · doi:10.3390/ph14080765 · Cited by 21
CRISPR Toolbox for Genome Editing in Dictyostelium
Yamashita, K., Iriki, H., Kamimura, Y., & Muramoto, T. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.721630 · Cited by 23
“The Cas9 nuclease is guided by a sgRNA for target-site recognition and then generates a DNA double-strand break (DSB) using two nuclease domains (RuvC and HNH) (Cong et al., 2013; Mali et al., 2013; Ran et al., 2013).”
Method
A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture
Karpov, D. S., Карпов, В. Л., Рафаиловна, К. Р., Demidova, N. A., & Aa, K. (2019) · Molecular Biology · doi:10.1134/s0026893319010059 · Cited by 20
Current therapies for osteoarthritis and prospects of CRISPR-based genome, epigenome, and RNA editing in osteoarthritis treatment
Chen, Y., Luo, X. L., Kang, R., Cui, K., Ou, J., Zhang, X., & Liang, P. (2023) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2023.07.007 · Cited by 23
The Application of CRISPR-Cas9 Genome Editing in Caenorhabditis elegans
Xu, S. (2015) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2015.06.005 · Cited by 19
“Consequently, 18 use of these Cas9 nickases significantly increased the efficiency of HDR at the expense of NHEJ (Cong et 19 al., 2013; Mali et al., 2013c) compared to the wild-type Cas9.”
BackgroundInfluential
Applications of CRISPR genome editing technology in drug target identification and validation
Lu, Q., Livi, G. P., Modha, S. S., Yusa, K., Macarrón, R., & Dow, D. J. (2017) · Expert Opinion on Drug Discovery · doi:10.1080/17460441.2017.1317244 · Cited by 21
“For the CRISPRa reaction, transcriptional activation by dCas9 fused with a transcription activator such as VP64 is inefficient compared with multiple gRNAs targeting the same gene [30-32].”
Method
Emerging applications of genome-editing technology to examine functionality of GWAS-associated variants for complex traits
Smith, A. J. P., Deloukas, P., & Munroe, P. B. (2018) · Physiological Genomics · doi:10.1152/physiolgenomics.00028.2018 · Cited by 21
Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs
Wang, Y., Zhao, J., Duan, N., Liu, W., Zhang, Y., Zhou, M., Hu, Z., Feng, M., Liu, X., Wu, L., et al. (2018) · International Journal of Molecular Sciences · doi:10.3390/ijms19103035 · Cited by 20
Applications of CRISPR systems in respiratory health: Entering a new ‘red pen’ era in genome editing
Moses, C., & Kaur, P. (2019) · Respirology · doi:10.1111/resp.13527 · Cited by 22
Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells
Petazzi, P., Torres-Ruiz, R., Fidanza, A., Roca-Ho, H., Gutiérrez‐Agüera, F., Castaño, J., Rodríguez, S., de la Guardia, R. D., López‐Millán, B., Bigas, A., et al. (2020) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2020.02.009 · Cited by 20
Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics
Galizi, R., Duncan, J. N., Rostain, W., Quinn, C. M., Storch, M., Kushwaha, M., & Jaramillo, A. (2020) · The CRISPR Journal · doi:10.1089/crispr.2020.0029 · Cited by 20
Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster
Waters, A. J., Capriotti, P., Gaboriau, D. C. A., Papathanos, P. A., & Windbichler, N. (2018) · Scientific Reports · doi:10.1038/s41598-018-31433-2 · Cited by 20
Induced Pluripotency and Gene Editing in Disease Modelling: Perspectives and Challenges
Seah, S., Farran, C. A. E., Warrier, T., Xu, J., & Loh, Y. (2015) · International Journal of Molecular Sciences · doi:10.3390/ijms161226119 · Cited by 21
Optogenetics and CRISPR: A New Relationship Built to Last
Mathony, J., Hoffmann, M. D., & Niopek, D. (2020) · Methods in molecular biology · doi:10.1007/978-1-0716-0755-8_18 · Cited by 20
Sophisticated CRISPR/Cas tools for fine-tuning plant performance
Capdeville, N., Merker, L., Schindele, P., & Puchta, H. (2020) · Journal of Plant Physiology · doi:10.1016/j.jplph.2020.153332 · Cited by 21
Potential Therapeutic Targeting of lncRNAs in Cholesterol Homeostasis
Ye, W.-C., Huang, S.-F., Hou, L.-J., Long, H.-J., Yin, K., Hu, C. Y., & Zhao, G.-J. (2021) · Frontiers in Cardiovascular Medicine · doi:10.3389/fcvm.2021.688546 · Cited by 19
A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells
Park, S. B., Uchida, T., Tilson, S. G., Hu, Z., Ma, C. D., Leek, M., Eichner, M., Hong, S. G., & Liang, T. J. (2022) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2022.04.013 · Cited by 18

Authors: Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M.
Affiliations: Harvard University (US)
Corresponding author: Church, George M
ORCID: Mali, Prashant
Aach, John
Stranges, P Benjamin
Esvelt, Kevin M
Moosburner, Mark
Kosuri, Sriram
Yang, Luhan
Church, George M
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2013-08-01
Volume: 31
Issue: 9
Pages: 833-838
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt.2675
PMC: PMC3818127
OpenAlex: W2026520402
Cited by: 1,752
References: 30
Citations by year: 2026: 33 · 2025: 45 · 2024: 71 · 2023: 83 · 2022: 122 · 2021: 119 · 2020: 125 · 2019: 159 · 2018: 154 · 2017: 190 · 2016: 231 · 2015: 175 · 2014: 209 · 2013: 36
FWCI: 97.27
Type: journal article
Language: en
Open access: green
europepmc.org
Licence: www.springer.com/tdm (tdm)
Locations: Nature Biotechnology (publishedVersion)
PubMed (publishedVersion)
Europe PMC (PubMed Central) (acceptedVersion)
PubMed Central (acceptedVersion)
Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
eScholarship (California Digital Library) (submittedVersion)
Funders: National Human Genome Research Institute (P50HG005550)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords: CRISPR/Cas9, transcriptional activation, single guide RNA, off-target binding, target specificity, TAL effectors, genome editing, target mismatch
MeSH terms: Base Sequence, Deoxyribonuclease I, Genetic Engineering, Humans, Models, Genetic, Molecular Sequence Data, Trans-Activators, HEK293 Cells, RNA, Small Untranslated, CRISPR-Associated Proteins

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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

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