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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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“To facilitate the design of primers, we generated primer sequences for the ,190,000 previously described specific gRNA-targetable sequences [11] (Table S1).”
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“PAM is a 3-base pair (NGG) sequence present near the target site and is necessary for binding and Cas9-induced DNA editing [52].”
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CRISPR–Cas9 in genome editing: Its function and medical applications
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“This system significantly reduces the off‐target cleavage through the performance of Cas9n is also slightly reduced compared to the wild type of the Cas9 protein (Mali et al., 2013).”
BackgroundPersonalized therapeutic strategies for patients with retinitis pigmentosa
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“Using a programmable short (17 -- 24 nucleotide) guide RNA (gRNA), Cas9 can then be directed to essentially any target site in the genome containing a 2 -- 4 nucleotide protospacer-adjacent motif sequence and induce double-strand breaks (DSBs) in the DNA (Figure 1) [48-50].”
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“…single strand break-mediated HDR efficiency is low, although recent reports have demonstrated that the combination of two nickases allows for the induction of off-set double strand breaks with 5′ overhang and the stimulation of template-mediated HDR events (Mali et al. 2013a; Ran et al. 2013).”
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BackgroundImproved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination
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Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells
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“In order to adapt the Cas9 nuclease for targeted gene activation, we and others have fused transcriptional activation domains to a Cas9 mutant in which the nuclease activity has been abolished [6-11].”
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New advances in CRISPR/Cas-mediated precise gene-editing techniques
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“…(nCas9) if they only cut one DNA strand (Cong et al., 2013). dCas9 and nCas9 have been used to reduce observed off-target effects (Ran et al., 2013; Mali et al., 2013) and as a chassis onto which to fuse other enzymes, such as deaminases (Gaudelli et al., 2017; Kim et al., 2017b; Komor et al.,…”
Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression
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“The large sequence space of possible protospacers makes it challenging to determine the positions that are most important for DNA binding (56, 120).”
BackgroundMethodInfluentialGenome editing strategies: potential tools for eradicating HIV-1/AIDS
Khalili, K., Kaminski, R., Gordon, J., Cosentino, L., & Hu, W. (2015) · Journal of NeuroVirology · doi:10.1007/s13365-014-0308-9 · Cited by 51
“However, the paired nickase approach requires two single monomeric nickases that may induce potential off-target indel mutations through an undefined mechanism (Mali et al. 2013a; Ran et al. 2013; Tsai et al. 2014).”
BackgroundMethodInfluentialCRISPR/Cas9: A Practical Approach in Date Palm Genome Editing
Sattar, M. N., Iqbal, Z., Tahir, M. N., Shahid, M. S., Khurshid, M., Al-Khateeb, A. A., & Al-Khateeb, S. A. (2017) · Frontiers in Plant Science · doi:10.3389/fpls.2017.01469 · Cited by 47
“Cleaving the target DNA sequence by pairing the two nickases can generate double stranded sticky ends break (Mali et al., 2013; Ran et al., 2013).”
BackgroundIn Planta Processing of the SpCas9–gRNA Complex
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“Extra nucleotides added to the 50 and 30 end of a gRNA have a negative effect on gRNA function (Hsu et al. 2013, Mali et al. 2013).”
BackgroundCRISPR/Cas9: the Jedi against the dark empire of diseases
Khan, S., Mahmood, M. S., Rahman, S. U., Zafar, H., Khan, S. H., khan, Z., & Ahmad, A. (2018) · Journal of Biomedical Science · doi:10.1186/s12929-018-0425-5 · Cited by 49
“The fusion of dCas9 with a transcription activator VP64 can result in the activation of a reporter gene [26, 27].”
Initiation of homologous recombination at DNA nicks
Maizels, N., & Davis, L. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky588 · Cited by 50
Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance
Babu, K., Amrani, N., Jiang, W., Yogesha, S. D., Nguyen, R., Qin, P. Z., & Rajan, R. (2019) · Biochemistry · doi:10.1021/acs.biochem.8b01241 · Cited by 47
Baba, M., Yokoyama, K., Seiriki, K., Naka, Y., Matsumura, K., Kondo, M., Yamamoto, K., Hayashida, M., Kasai, A., Ago, Y., et al. (2019) · Neuropsychopharmacology · doi:10.1038/s41386-019-0441-5 · Cited by 49
“We utilized the Cas9 nickase (D10A) mutant to reduce off-target mutagenesis [32, 33].”
MethodComparative analysis of dCas9-VP64 variants and multiplexed guide RNAs mediating CRISPR activation
Omachi, K., & Miner, J. H. (2022) · PLoS ONE · doi:10.1371/journal.pone.0270008 · Cited by 48
“Lenti-EF1α-scFv-VP64_Blast was generated by fusing scFv-sfGFP-GB1 from pHRdSV40_scFv_GCN4_sfGFP_p65-hsf1_GB1_NLS (Addgene plasmid #79372, a gift from George Church) [19] and VP64 from lenti dCAS-VP64_Blast (Addgene plasmid #61425) using Gibson Assembly and inserting into the backbone of lenti dCAS-VP64_Blast (Addgene…”
MethodThe trans-regulatory landscape of gene networks in plants
Hummel, N. F. C., Zhou, A., Li, B., Markel, K., Ornelas, I. J., & Shih, P. M. (2023) · Cell Systems · doi:10.1016/j.cels.2023.05.002 · Cited by 45
Performance of the Cas9 Nickase System in Drosophila melanogaster
Ren, X., Yang, Z., Mao, D., Chang, Z., Qiao, H.-H., Wang, X., Sun, J., Hu, Q., Cui, Y., Liu, L.-P., et al. (2014) · G3 Genes|Genomes|Genetics · doi:10.1534/g3.114.013821 · Cited by 40
“In addition, we have observed that one sgRNA does not trigger mutations in Cas9 nickase transgenic flies, suggesting that the Cas9 nickase system significantly prevents offtarget effects.”
BackgroundInfluentialA CRISPR method for genome engineering
Wilkinson, R. A., & Wiedenheft, B. (2014) · F1000Prime Reports · doi:10.12703/p6-3 · Cited by 46
Genome engineering for breaking barriers in lignocellulosic bioethanol production
Ulaganathan, K., Goud, S., Reddy, M., & Ulaganathan, K. (2017) · Renewable and Sustainable Energy Reviews · doi:10.1016/j.rser.2017.01.028 · Cited by 45
The Chromatin Structure Differentially Impacts High-Specificity CRISPR-Cas9 Nuclease Strategies
Chen, X., Liu, J., Janssen, J. M., & Gonçalves, M. A. F. V. (2017) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2017.08.005 · Cited by 47
Programmable Molecular Scissors: Applications of a New Tool for Genome Editing in Biotech
Saha, S. K., Saikot, F. K., Rahman, M. S., Jamal, M. A. H. M., Rahman, S. M. K., Islam, S. M. R., & Kim, K. (2018) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2018.11.016 · Cited by 48
Induced Methylation in Plants as a Crop Improvement Tool: Progress and Perspectives
Mercé, C., Bayer, P. E., Fernandez, C. G. T., Batley, J., & Edwards, D. (2020) · Agronomy · doi:10.3390/agronomy10101484 · Cited by 46
Hoffmann, M. D., Mathony, J., zu Belzen, J. U., Harteveld, Z., Aschenbrenner, S., Stengl, C., Grimm, D., Correia, B. E., Eils, R., & Niopek, D. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa1198 · Cited by 45
“regulation of endogenous transcription (4,5), epigenetic reprogramming (6-8), DNA labeling (9,10) and base editing (11,12).”
BackgroundGenetically modified large animal models for investigating neurodegenerative diseases
Yang, W., Chen, X., Li, S., & Li, X.-J. (2021) · Cell & Bioscience · doi:10.1186/s13578-021-00729-8 · Cited by 45
CRISPR/Cas9 and Nanotechnology Pertinence in Agricultural Crop Refinement
Naik, B. J., Shimoga, G. D., Kim, S.-C., Manjulatha, M., Palem, R. R., Palem, R. R., Kumar, M., Kim, S., & Lee, S. (2022) · Frontiers in Plant Science · doi:10.3389/fpls.2022.843575 · Cited by 46
Optimized tuning of TALEN specificity using non-conventional RVDs
Juillerat, A., Pessereau, C., Dubois, G., Revol-Guyot, V., Maréchal, A., Valton, J., Daboussi, F., Poirot, L., Duclert, A., & Duchateau, P. N. (2015) · Scientific Reports · doi:10.1038/srep08150 · Cited by 44
CRISPRlnc: a manually curated database of validated sgRNAs for lncRNAs
Chen, W., Zhang, G., Li, J., Zhang, X., Huang, S., Xiang, S., Hu, X., & Liu, C. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky904 · Cited by 44
FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity
Cui, Z., Tian, R., Huang, Z., Jin, Z., Li, L., Liu, J., Huang, Z., Xie, H., Liu, D., Mo, H., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-29089-8 · Cited by 46
Richter, F. M., Fonfara, I., Gelfert, R., Nack, J., Charpentier, E., & Möglich, A. (2017) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2017.03.025 · Cited by 44
Emerging applications of gene editing technologies for the development of climate-resilient crops
Chavhan, R. L., Jaybhaye, S. G., Hinge, V. R., Deshmukh, A. S., Shaikh, U. S., Jadhav, P. K., Kadam, U. S., & Hong, J. C. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1524767 · Cited by 55
Park, A., Won, S. T., Pentecost, M., Wojciech, B., & Lee, B. (2014) · PLoS ONE · doi:10.1371/journal.pone.0095101 · Cited by 43
“Although we used the wild-type Cas9 endonuclease that catalyzes both nicks and doublestrand breaks (DSBs), the use of a mutant Cas9 that only nicks may be used to avoid DSB-mediated non-homologous end joining mutations in off-target sites [26,30,32].”
Comprehensive Protocols for CRISPR/Cas9‐based Gene Editing in Human Pluripotent Stem Cells
Santos, D. P., Kiskinis, E., Eggan, K. C., & Merkle, F. T. (2016) · Current Protocols in Stem Cell Biology · doi:10.1002/cpsc.15 · Cited by 45
“…in stem cells, many groups began showing that the technology could be used for generation of gene knock-out cell lines (Cong et al., 2013; Mali et al., 2013b), small targeted mutations (Miyaoka et al., 2014; Ran et al., 2013a), and knock-in stem cell lines (Mali et al., 2013a; Merkle et…”
BackgroundInfluentialGenome Editing of Monogenic Neuromuscular Diseases
Long, C., Amoasii, L., Bassel‐Duby, R., & Olson, E. N. (2016) · JAMA Neurology · doi:10.1001/jamaneurol.2016.3388 · Cited by 46
Making ends meet: targeted integration of DNA fragments by genome editing
Yamamoto, Y., & Gerbi, S. A. (2018) · Chromosoma · doi:10.1007/s00412-018-0677-6 · Cited by 49
“Off-target insertion has been minimized for CRISPR approaches such as by using a truncated sgRNA (Fu et al. 2014) or using a pair of Cas9s modified to act as nickases with staggered cuts on opposite DNA strands (Mali et al. 2013; Ran et al. 2013).”
BackgroundRaikwar, S. P., Kikkeri, N. S., Sakuru, R. C., Saeed, D., Zahoor, H., Premkumar, K., Mentor, S., Thangavel, R., Dubova, I., Ahmed, M. E., et al. (2019) · Journal of Neuroimmune Pharmacology · doi:10.1007/s11481-019-09849-y · Cited by 48
“…subsequently exploited to allow double strand breaks in the mammalian genome at specified locations that could be repaired by either nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) (Cong et al. 2013; Mali et al. 2013a; Mali et al. 2013b; Ran et al. 2013a; Sander and Joung 2014).”
BackgroundKulcsár, P. I., Tálas, A., Ligeti, Z., Krausz, S. L., & Welker, E. (2022) · Nature Communications · doi:10.1038/s41467-022-34527-8 · Cited by 46
“5 ng of both sgRNA coding plasmids was added); (2) in case of genomic base editing: base editor variant expression plasmid (190 ng) and sgRNA and BFP coding plasmid (83 ng); (3) in case of genomic prime editing: prime editor variant expression plasmid (222ng), pegRNA and BFP coding plasmid (55 ng), and second nicking…”
BackgroundMethodPrime editing primarily induces undesired outcomes in mice
Aida, T., Wilde, J. J., Yang, L., Hou, Y., Li, M., Xu, D., Lin, J., Qi, P., Lu, Z., & Feng, G. (2020) · doi:10.1101/2020.08.06.239723 · Cited by 43
“…requires induction of nicks on both DNA strands has also been utilized to induce DSBs and subsequent indels by zinc finger nucleases (Miller et al., 2007), transcription-activator-like effector nucleases (Miller et al., 2011), and CRISPR Cas9-D10A nickases (Mali et al., 2013; Ran et al., 2013).”
MethodResultFujita, T., & Fujii, H. (2014) · PLoS ONE · doi:10.1371/journal.pone.0103084 · Cited by 42
Personalised genome editing – The future for corneal dystrophies
Moore, T., Christie, K. A., Marshall, J., & Nesbit, M. A. (2018) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2018.01.004 · Cited by 44
Yamamoto, A., Ishida, T., Yoshimura, M., Kimura, Y., & Sawa, S. (2019) · Plant and Cell Physiology · doi:10.1093/pcp/pcz118 · Cited by 42
Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes
Zhang, L., Rube, H. T., Vakulskas, C. A., Behlke, M. A., Bussemaker, H. J., & Pufall, M. A. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa231 · Cited by 45
“The DNA-binding specificity of dSpCas9::Nanog (3,8,49) was measured by Spec-seq.”
MethodRegulating CRISPR/Cas9 Function through Conditional Guide RNA Control
Brown, W., Zhou, W., & Deiters, A. (2020) · ChemBioChem · doi:10.1002/cbic.202000423 · Cited by 42
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
Self-citation
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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