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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
Open accessAbstract
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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“The markedly high efficiency of CRISPR/Cas systems coupled with the above-mentioned advantages over other methods justify the CRISPR “epidemic” that the scientific community experienced in 2013 [62,81,100,101].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [101]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“Scientists engineered a double nicking scheme featuring a pair of Cas9n targeting opposite strands where neighbouring gRNA targets are offset by a certain number of base pairs [101].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [101]
In the citing paper
“Consistent with a previous report 19 , this suggests that TALENs can tolerate two mismatches in vivo.”
From TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis (2014) · cites this paper as [19]
In the citing paper
“However, it has been recently shown that CRISPR/Cas9 system can be highly active even when mismatches occur between gRNA and their DNA targets”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [92]
In the citing paper
“and off-target mutations occur at endogenous loci in human cells with high frequency [90,91,92].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [92]
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“For example, Cas9/gRNA seems to have considerable offtarget effects in cultured human cells (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013) and in several organisms (Fujii et al., 2013; Li et al., 2013; Shan et al., 2013).”
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A CRISPR-based approach for targeted DNA demethylation
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Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants
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“Paired nicks generated by nCas9 have the potential to substantially increase cleavage specificity, given that two gRNAs are required per DSB (Mali et al., 2013a; Ran et al., 2013), and we have determined that nCas9-generated paired nicks efficiently induce translocations.”
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“To test this possibility, two pairs of nickase-expressing plasmids (Mali et al. 2013; Ran et al. 2013a) targeting the p53 gene and the Rb1 gene separately were constructed and cotransfected into the OctaKO cells together with the RFP/GFP-HygR reporter plasmid (Fig.”
CRISPR-Cas9 system: A new-fangled dawn in gene editing
Gupta, D., Bhattacharjee, O., Mandal, D., Sen, M. K., Dey, D., Dasgupta, A., Kazi, T. A., Gupta, R. K., Sinharoy, S., Acharya, K., et al. (2019) · Life Sciences · doi:10.1016/j.lfs.2019.116636 · Cited by 351
CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool
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“This conclusion is further supported by the observation that sgRNAs targeting the y, w, v, and e genes do not induce off-target mutations in genomic DNA carrying three mismatches (Figure 1D and Figure S1B), and is in line with recent studies in mammalian cells (Hsu et al., 2013; Mali et al., 2013; Wang et al., 2014).”
BackgroundResultInfluentialUniversal CARs, universal T cells, and universal CAR T cells
Zhao, J., Lin, Q., Song, Y., & Liu, D. (2018) · Journal of Hematology & Oncology · doi:10.1186/s13045-018-0677-2 · Cited by 324
Targeted genomic rearrangements using CRISPR/Cas technology
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A Mouse Geneticist’s Practical Guide to CRISPR Applications
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“Site-specific nicks are predominantly repaired by high fidelity base excision repair pathway (BER) or homology driven repair (HDR) events (Figure 1), but rarely by NHEJ (Cong et al. 2013; Mali et al. 2013a).”
BackgroundCRISPR/Cas9-based engineering of the epigenome
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Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity
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“…al., 2015; Tsai et al., 2015; Wang et al., 2015) and are important complements to biased off-target detectionmethods based on in silico prediction and targeted sequencing (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013) (see Unbiased Genomewide Off-Target Detection).”
BackgroundMethodInfluentialIsolation of single-base genome-edited human iPS cells without antibiotic selection
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Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice
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A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans
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“…(Bassett et al. 2013; Chang et al. 2013; Cho et al. 2013a; Cong et al. 2013; Dicarlo et al. 2013; Dickinson et al. 2013; Feng et al. 2013, 2014; Friedland et al. 2013; Gratz et al. 2013; Jiang et al. 2013; Mali et al. 2013b; Wang et al. 2013; Ma et al. 2014; Yu et al. 2014; Zhou et al. 2014).”
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“…Cas9 (Qi et al., 2013), which can modulate transcription in bacteria or eukaryotes either directly (Qi et al., 2013; Bikard et al., 2013) or through an incorporated effector domain (Gilbert et al., 2013; Mali et al., 2013a; Konermann et al., 2013; Maeder et al., 2013; Perez-Pinera et al., 2013).”
BackgroundMethodResultInfluentialMultiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts
Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D., & Platt, R. J. (2019) · Nature Methods · doi:10.1038/s41592-019-0508-6 · Cited by 290
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“SpyCas9 nickases (nCas9s) have been used to induce homology-directed repair (HDR) and to improve genome editing specificity via DSB induction by dual nickases (Mali et al., 2013a; Ran et al., 2013).”
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“…and H840A; henceforth referred to as dCas9) that lacks endonuclease activity but can still be recruited by sgRNA(s) (Jinek et al., 2012) has recently been used to target genes in mammalian cells for transcriptional activation (Perez-Pinera et al., 2013a,b; Maeder et al., 2013; Mali et al., 2013).”
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Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9
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Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease
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CRISPR/cas systems redefine nucleic acid detection: Principles and methods
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Genome modification by CRISPR /Cas9
Ma, Y., Zhang, L., & Huang, X. (2014) · FEBS Journal · doi:10.1111/febs.13110 · Cited by 304
The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops
Khatodia, S., Bhatotia, K., Passricha, N., Khurana, S. M. P., & Tuteja, N. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.00506 · Cited by 267
“…been used into a paired nickase system with two different gRNA to extend the number of specifically recognized bases for target cleavage, which has improved the specificity and helped mitigate the off-target phenomena (Cong et al., 2013; Mali et al., 2013; Ran et al., 2013; Fauser et al., 2014).”
BackgroundMethodA Broad-Spectrum Inhibitor of CRISPR-Cas9
Harrington, L. B., Doxzen, K. W., Ma, E., Liu, J., Knott, G. J., Edraki, A., García, B., Amrani, N., Chen, J. S., Cofsky, J. C., et al. (2017) · Cell · doi:10.1016/j.cell.2017.07.037 · Cited by 259
“…is inhibited and Cas9 is caused to dimerize. this occurs in nature, it is an enticing possibility that AcrIIC1 can be employed in gene-editing applications to obviate the need to generate separate dCas9 enzymes for gene-regulatory purposes (Gilbert et al., 2014; Mali et al., 2013; Qi et al., 2013).”
BackgroundMiyaoka, Y., Berman, J. R., Cooper, S., Mayerl, S. J., Chan, A. H., Zhang, B., Karlin-Neumann, G. A., & Conklin, B. R. (2016) · Scientific Reports · doi:10.1038/srep23549 · Cited by 277
“• NWT+ = number of droplets in the clusters labeled “WT+ ” (green).”
InfluentialReal-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A., & Ha, T. (2016) · Nature Communications · doi:10.1038/ncomms12778 · Cited by 269
The roles of CRISPR–Cas systems in adaptive immunity and beyond
Barrangou, R. (2015) · Current Opinion in Immunology · doi:10.1016/j.coi.2014.12.008 · Cited by 276
Long, C., Li, H. Z., Tiburcy, M., Rodríguez-Caycedo, C., Kyrychenko, V., Zhou, H., Zhang, Y., Min, Y.-L., Shelton, J. M., Mammen, P. P. A., et al. (2018) · Science Advances · doi:10.1126/sciadv.aap9004 · Cited by 261
Advances in therapeutic CRISPR/Cas9 genome editing
Savić, N., & Schwank, G. (2016) · Translational Research · doi:10.1016/j.trsl.2015.09.008 · Cited by 257
Multi‐input CRISPR/Cas genetic circuits that interface host regulatory networks
Nielsen, A. A. K., & Voigt, C. A. (2014) · Molecular Systems Biology · doi:10.15252/msb.20145735 · Cited by 259
“…regarding off-target Cas9 interactions and several efforts seeking to reduce it (Cradick et al , 2013; Fu et al , 2013, 2014; Hsu et al , 2013; Mali et al , 2013; Pattana-yak et al , 2013; Ran et al , 2013; Guilinger et al , 2014; Kuscu et al , 2014; Tsai et al , 2014; Wu et al , 2014), this…”
BackgroundOrmond, K. E., Mortlock, D. P., Scholes, D. T., Bombard, Y., Brody, L. C., Faucett, W. A., Garrison, N. A., Hercher, L., Isasi, R., Middleton, A., et al. (2017) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2017.06.012 · Cited by 252
Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing
Robert, F., Barbeau, M., Ethier, S. D., Dostie, J., & Pelletier, J. (2015) · Genome Medicine · doi:10.1186/s13073-015-0215-6 · Cited by 268
Investigating human disease using stem cell models
Sterneckert, J. L., Reinhardt, P., & Schöler, H. R. (2014) · Nature Reviews Genetics · doi:10.1038/nrg3764 · Cited by 262
Cas9-mediated targeting of viral RNA in eukaryotic cells
Price, A. A., Sampson, T. R., Ratner, H. K., Grakoui, A., & Weiss, D. S. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1422340112 · Cited by 256
Partial DNA-guided Cas9 enables genome editing with reduced off-target activity
Yin, H., Song, C.-Q., Suresh, S., Kwan, S.-Y., Wu, Q., Walsh, S., Ding, J., Bogorad, R. L., Zhu, L. J., Wolfe, S. A., et al. (2018) · Nature Chemical Biology · doi:10.1038/nchembio.2559 · Cited by 240
Genome Editing with Engineered Nucleases in Plants
Osakabe, Y., & Osakabe, K. (2014) · Plant and Cell Physiology · doi:10.1093/pcp/pcu170 · Cited by 253
“The CRISPR/Cas9 system has been used successfully to cleave target DNA and introduce mutations in eukaryotic cells, including human cells (Cong et al. 2013, Mali et al. 2013a, Mussolino and Cathomen 2013) and plants (Li et al. 2013, Nekrasov et al. 2013, Shan et al. 2013b).”
BackgroundMethodInfluentialKhan, S. H. (2019) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2019.02.027 · Cited by 254
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells
Lee, C. M., Cradick, T. J., & Bao, G. (2016) · Molecular Therapy · doi:10.1038/mt.2016.8 · Cited by 250
“10 20 Current strategies to reduce CRISPR-Cas9 off-target effects include the use of partially inactivated Cas9 nickases 21,22 and a catalytically dead Cas9 fused to the FokI endonuclease domain, 23–25 both of which require the design and delivery of two gRNAs.”
Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC
Kwon, D. Y., Zhao, Y.-T., Lamonica, J. M., & Zhou, Z. (2017) · Nature Communications · doi:10.1038/ncomms15315 · Cited by 246
Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned
Morgan, R. A., Gray, D., Lomova, A., & Kohn, D. B. (2017) · Cell stem cell · doi:10.1016/j.stem.2017.10.010 · Cited by 252
Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression
Luo, M. L., Mullis, A. S., Leenay, R. T., & Beisel, C. L. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku971 · Cited by 247
Review of CRISPR/Cas9 sgRNA Design Tools
Cui, Y., Xu, J., Cheng, M., Liao, X., & Peng, S. (2018) · Interdisciplinary Sciences Computational Life Sciences · doi:10.1007/s12539-018-0298-z · Cited by 253
Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM
Wong, A. S. L., Choi, G. C. G., Cui, C. H., Pregernig, G., Milani, P., Adam, M., Perli, S. D., Kazer, S. W., Gaillard, A., Hermann, M., et al. (2016) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1517883113 · Cited by 240
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
Live 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
Colasante, G., Lignani, G., Brusco, S., Berardino, C. D., Carpenter, J. C., Giannelli, S. G., Valassina, N., Bido, S., Ricci, R., Castoldi, V., et al. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.08.018 · Cited by 246
Nucleosome breathing and remodeling constrain CRISPR-Cas9 function
Isaac, R. S., Jiang, F., Doudna, J. A., Lim, W. A., Narlikar, G. J., & Almeida, R. (2016) · eLife · doi:10.7554/elife.13450 · Cited by 234
“…tool for genome engineering, gene regulation, and live imaging is a remarkable achievement with profound impact in a wide variety of research fields and applications ( Makarova et al., 2015 ; Doudna and Charpentier, 2014 ; Cong et al., 2013 ; Jinek et al., 2012 ; 2013 ; Mali et al., 2013 ).”
InfluentialSingle Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects
Gao, Y., Wu, H., Wang, Y., Liu, X., Chen, L., Li, Q., Cui, C., Liu, X., Zhang, J., & Zhang, Y. (2017) · Genome biology · doi:10.1186/s13059-016-1144-4 · Cited by 246
“These strategies include the use of double nickase [19, 20], short sgRNAs [50], and a fusion”
BackgroundMethodCRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice
Yin, X., Biswal, A. K., Dionora, J., Perdigon, K. M., Balahadia, C. P., Mazumdar, S., Chater, C., Lin, H., Coe, R. A., Kretzschmar, T., et al. (2017) · Plant Cell Reports · doi:10.1007/s00299-017-2118-z · Cited by 241
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
Xu, R., Li, H., Qin, R., Li, J., Qiu, C.-H., Yang, Y.-C., Ma, H., Li, L., Wei, P., & Yang, J.-B. (2015) · Scientific Reports · doi:10.1038/srep11491 · Cited by 227
A CRISPR view of developmentPreprint
Harrison, M. M., Jenkins, B., O’Connor-Giles, K. M., & Wildonger, J. (2014) · Genes & Development · doi:10.1101/gad.248252.114 · Cited by 234
“…genes simultaneously has been carried out in cultured mammalian cells as well as genetic model organisms such as mice, zebrafish, and Arabidopsis (Chang et al. 2013; Cong et al. 2013; Feng et al. 2013; Hwang et al. 2013; Li et al. 2013a; Mali et al. 2013c; Mao et al. 2013; Wang et al. 2013a).”
BackgroundInfluentialCas9-chromatin binding information enables more accurate CRISPR off-target prediction
Singh, R., Kuscu, C., Quinlan, A., Qi, Y., & Adli, M. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv575 · Cited by 217
A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
O'Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., & Segal, D. J. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv137 · Cited by 219
Weissman, D. (2015) · Expert Review of Vaccines · doi:10.1586/14760584.2015.973859 · Cited by 228
“The RNA tags identify the site for cutting [154,155].”
BackgroundJasin, M., & Haber, J. E. (2016) · DNA repair · doi:10.1016/j.dnarep.2016.05.001 · Cited by 229
“In addition to using enhanced Cas9s, another route is to reducing off-target effects is to use nickases to create two nicks on opposite strands to generate a DSB [182, 183].”
Background
Metadata
- 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
- Venue
- Nature Biotechnology
- Published
- 2013-08-01
- Volume
- 31
- Issue
- 9
- Pages
- 833-838
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- 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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