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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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In the citing paper
“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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Cites
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Exploiting CRISPR–Cas immune systems for genome editing in bacteria
Barrangou, R., & van Pijkeren, J. (2015) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2015.10.003 · Cited by 75
“In particular, variants of Cas9 in which both domains have been inactivated, generated a deactivated Cas9 (dCas9), constitute DNA binding proteins that can be used as transcriptional effectors when targeting promoter regions, either as repressors that block RNA polymerases, or fused to activators that enhance transcri…”
BackgroundDetection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique
Zhang, B., Wang, Q., Xu, X., Xia, Q., Long, F., Li, W., Shui, Y., Xia, X., & Wang, J. (2018) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-018-0873-5 · Cited by 72
CRISPR/Cas9 Nuclease-Mediated Gene Knock-In in Bovine-Induced Pluripotent Cells
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“Potential targets of CRISPR sgRNAs were identified as previously described (Mali et al., 2013) [29].”
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Zhang, D., Li, Z., & Li, J.-F. (2016) · Journal of Genetics and Genomics · doi:10.1016/j.jgg.2016.03.001 · Cited by 67
“Pronounced activation had to rely on many sgRNAs targeting to the same promoter (Mali et al., 2013a; Perez-Pinera et al., 2013; Lowder et al., 2015), which dramatically reduces the total number of genes that can be activated concurrently due to M AN US CR IP T AC CE PT ED 16 the cloning and…”
BackgroundMethodInfluentialSingle and multiple gene knockouts by CRISPR–Cas9 in maize
Doll, N. M., Gilles, L. M., Gérentes, M.-F., Richard, C., Just, J., Fierlej, Y., Borrelli, V. M. G., Gendrot, G., Ingram, G. C., Rogowsky, P. M., et al. (2019) · Plant Cell Reports · doi:10.1007/s00299-019-02378-1 · Cited by 71
Class 2 CRISPR–Cas RNA-guided endonucleases: Swiss Army knives of genome editing
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Genome Engineering of Eubacterium limosum Using Expanded Genetic Tools and the CRISPR-Cas9 System
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CRISPR-based metabolic pathway engineering
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“Paired nickases generate two single-strand breaks or nicks on different DNA strands, resulting in composite DSB and doubling the specificity of genome editing (Cho et al. 2014; Kim et al. 2012; Mali et al. 2013; Ran et al. 2013).”
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Sakuma, T., Masaki, K., Abe‐Chayama, H., Mochida, K., Yamamoto, T., & Chayama, K. (2016) · Genes to Cells · doi:10.1111/gtc.12437 · Cited by 72
“To evaluate their DSB-inducing activity, we carried out a human cellbased reporter assay known as single-strand annealing (SSA) (Ochiai et al. 2010; Sakuma et al. 2013).”
MethodSun, Z., Wang, M., Han, S., Ma, S., Zou, Z. V., Ding, F., Li, X., Li, L., Tang, B., Wang, H., et al. (2018) · Scientific Reports · doi:10.1038/s41598-018-32024-x · Cited by 70
Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control
Xu, T., Li, Y., Nostrand, J. D. V., He, Z., & Zhou, J. Z. (2014) · Applied and Environmental Microbiology · doi:10.1128/aem.03786-13 · Cited by 71
Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes
Pattanayak, V., Guilinger, J. P., & Liu, D. R. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00003-9 · Cited by 70
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Towards a new era in medicine: therapeutic genome editing
Porteus, M. H. (2015) · Genome Biology · doi:10.1186/s13059-015-0859-y · Cited by 69
Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9
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“As for CRISPR/ Cas9, we chose the pX335 CRISPR/Cas9 expression vector, which uses a double-nicking strategy, by which the occurrence of a double-strand break (DSB) becomes more specific and accurate (Mali et al., 2013; Ran et al., 2013).”
MethodSynthetic Biological Circuits within an Orthogonal Central Dogma
Costello, A., & Badran, A. H. (2020) · Trends in biotechnology · doi:10.1016/j.tibtech.2020.05.013 · Cited by 71
CRISPR–Cas system: a powerful tool for genome engineering
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“RNA-guided Cas9 activity creates site-specific DSBs, which are then repaired by either NHeJ or HDR, the sequence at the repair site can be modified or new genetic information inserted (Cong et al. 2013; Mali et al. 2013c; Cho et al. 2013) (Fig.”
BackgroundInfluentialRNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?
Gasiūnas, G., & Šikšnys, V. (2013) · Trends in Microbiology · doi:10.1016/j.tim.2013.09.001 · Cited by 70
Jaskula-Ranga, V., Wahlin, K. J., Maruotti, J., & Zack, D. J. (2014) · Nature Communications · doi:10.1038/ncomms5516 · Cited by 74
Genetic and Genomic Tools for the Marine AnnelidPlatynereis dumerilii
Zantke, J., Bannister, S. C., Rajan, V. B. V., Raible, F., & Tessmar‐Raible, K. (2014) · Genetics · doi:10.1534/genetics.112.148254 · Cited by 72
“…technologies currently used for custom nuclease-based genome engineering are zinc finger nucleases (ZFNs) (Kim et al. 1996), TALENs (Cermak et al. 2011), and Cas9/clustered regularly interspaced short palindromic repeats (CRISPR) systems (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013a).”
BackgroundMethodDong, F., Xie, K., Chen, Y., Yang, Y., & Mao, Y. (2016) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2016.11.129 · Cited by 75
“The nuclease defective Cas9 (dCas9) and gRNA were also engineered to control ex pression of targeted genes [8-11] and labeling specific loci of the chromosomes [12] in vivo.”
APOBEC: From mutator to editor
Yang, B., Li, X., Lei, L., & Chen, J. (2017) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2017.04.009 · Cited by 70
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“To reduce off-target effects of single gRNA-guided Cas9 nuclease, a previously reported “paired nicking” strategy was employed, in which two sgRNAs targeted to adjacent sites on opposite DNA strands [44, 50], as two sgRNA, driven by U6 promoter, were designed to target the first intron of the Rosa26 gene as reported (…”
MethodVaccine and antibody production in plants: developments and computational tools
Dubey, K. K., Luke, G. A., Knox, C. M., Kumar, P., Pletschke, B. I., Singh, P. K., & Shukla, P. (2018) · Briefings in Functional Genomics · doi:10.1093/bfgp/ely020 · Cited by 69
What has single-cell RNA-seq taught us about mammalian spermatogenesis?
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High-Throughput Approaches to Pinpoint Function within the Noncoding Genome
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“Extensive characterization of CRISPR-Cas systems has culminated in its repurposing as a facile platform for eukaryotic genome editing (Cong et al., 2013; Mali et al., 2013a), which has spurred a revolution in biology and medicine (Barrangou and Doudna, 2016).”
InfluentialFine-Tuning Next-Generation Genome Editing Tools
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“Paired nickases generate two single-strand breaks or nicks on different DNA strands, resulting in a composite DSB and doubling the specificity of genome editing [12, 33, 39, 40].”
The CRISPR/Cas9 genome editing methodology as a weapon against human viruses.
White, M. K., Hu, W., & Khalili, K. (2015) · PubMed · Cited by 66
Improved design and analysis of CRISPR knockout screens
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CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells
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Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections
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Resources for Functional Genomics Studies in Drosophila melanogaster
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Expression of CRISPR/Cas single guide RNAs using small tRNA promoters
Mefferd, A. L., Kornepati, A. V. R., Bogerd, H. P., Kennedy, E. M., & Cullen, B. R. (2015) · RNA · doi:10.1261/rna.051631.115 · Cited by 70
“Previous work has focused on using the U6 Pol III promoter to drive sgRNA transcription (Cong et al. 2013; Mali et al. 2013b; Kennedy et al. 2014; Swiech et al. 2015).”
InfluentialYin, C., Zhang, T., Li, F., Yang, F., Putatunda, R., Young, W., Khalili, K., Hu, W., & Zhang, Y. (2016) · AIDS · doi:10.1097/qad.0000000000001079 · Cited by 67
In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies
Yanik, M., Müller, B., Song, F., Gáll, J., Wagner, F., Wende, W., Lorenz, B., & Stieger, K. (2016) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2016.09.001 · Cited by 73
Evolving AAV-delivered therapeutics towards ultimate cures
He, X., Urip, B. A., Zhang, Z., Ngan, C. C., & Feng, B. (2021) · Journal of Molecular Medicine · doi:10.1007/s00109-020-02034-2 · Cited by 70
“The sgRNA in CRISPR system can tolerate minor mismatches to guide DNA cleavage at an off-target site [193].”
BackgroundEfficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system
Fujii, W., Onuma, A., Sugiura, K., & Naito, K. (2014) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2014.01.141 · Cited by 66
Stem cells for investigation and treatment of inherited retinal disease
Tucker, B. A., Mullins, R. F., & Stone, E. M. (2014) · Human Molecular Genetics · doi:10.1093/hmg/ddu124 · Cited by 65
Wattanapanitch, M., Damkham, N., Potirat, P., Trakarnsanga, K., Janan, M., U‐pratya, Y., Kheolamai, P., Klincumhom, N., & Issaragrisil, S. (2018) · Stem Cell Research & Therapy · doi:10.1186/s13287-018-0779-3 · Cited by 65
“The latter can be achieved via nickases that induce single-strand breaks [32, 33] or single-guide RNA (sgRNA)-guided catalytically inactive Cas9 (dCas9) fused to the FokI nuclease [34–36].”
BackgroundGenome editing in cardiovascular diseases
Mani, I. (2021) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2021.01.021 · Cited by 66
Gene-Editing Technologies and Applications in Legumes: Progress, Evolution, and Future Prospects
Baloğlu, M. C., Altunoğlu, Y. Ç., Baloglu, P., Yildiz, A. B., Türkölmez, N., & Çiftçi, Y. Ö. (2022) · Frontiers in Genetics · doi:10.3389/fgene.2022.859437 · Cited by 74
“Practically, the CRISPR/Cas9 system is designed for the replacement of the tracrRNA and crRNA molecules of the bacteria with the guide RNA (gRNA) (Cong et al., 2013; Mali et al., 2013).”
BackgroundThe iCRISPR Platform for Rapid Genome Editing in Human Pluripotent Stem Cells
Zhu, Z., González, F., & Huangfu, D. (2014) · Methods in Enzymology · doi:10.1016/b978-0-12-801185-0.00011-8 · Cited by 66
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BackgroundStrategies for precision modulation of gene expression by epigenome editing: an overview
Laufer, B. I., & Singh, S. M. (2015) · Epigenetics & Chromatin · doi:10.1186/s13072-015-0023-7 · Cited by 70
Nuclease‐mediated genome editing: At the front‐line of functional genomics technology
Sakuma, T., & Woltjen, K. (2014) · Development Growth & Differentiation · doi:10.1111/dgd.12111 · Cited by 70
Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design
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Maggio, I., Zittersteijn, H. A., Wang, Q., Liu, J., Janssen, J. M., Ojeda, I. T., van der Maarel, S. M., Lankester, A. C., Hoeben, R. C., & Gonçalves, M. A. F. V. (2020) · Gene Therapy · doi:10.1038/s41434-019-0119-y · Cited by 66
“20, herein named gRNA and gRNA, respectively, were previously described [34, 38].”
BackgroundMethodApplying CRISPR–Cas9 tools to identify and characterize transcriptional enhancers
Lopes, R., Korkmaz, G., & Agami, R. (2016) · Nature Reviews Molecular Cell Biology · doi:10.1038/nrm.2016.79 · Cited by 65
CRISPR-Cas9-Based Technology and Its Relevance to Gene Editing in Parkinson’s Disease
Rahman, M. U., Bilal, M., Shah, J. A., Kaushik, A. K., Teissèdre, P., & Kujawska, M. (2022) · Pharmaceutics · doi:10.3390/pharmaceutics14061252 · Cited by 65
Application of CRISPR/Cas9 genome editing to the study and treatment of disease
Pellagatti, A., Dolatshad, H., Valletta, S., & Boultwood, J. (2015) · Archives of Toxicology · doi:10.1007/s00204-015-1504-y · Cited by 65
“Other studies, in fact, demonstrated that Cas9 tolerates mismatches in a manner depending on their number, position and distribution (Pattanayak et al. 2013; Mali et al. 2013a; Hsu et al. 2013; Fu et al. 2013).”
BackgroundMethodInfluentialDesigned nucleases for targeted genome editing
Lee, J., Chung, J., Kim, H. M., Kim, S., & Kim, S. (2015) · Plant Biotechnology Journal · doi:10.1111/pbi.12465 · Cited by 70
Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing
Li, J., Hong, S., Chen, W., Zuo, E., & Yang, H. (2019) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2019.11.002 · Cited by 66
CRISPR-Based Synthetic Transcription Factors In Vivo: The Future of Therapeutic Cellular Programming
Pandelakis, M., Delgado, E., & Ebrahimkhani, M. R. (2020) · Cell Systems · doi:10.1016/j.cels.2019.10.003 · Cited by 63
“dCas9 gRNA can tolerate base extensions on two of its structural stem loops or its 3’ end, allowing for the addition of peptide recruiting aptamers (Mali et al., 2013).”
BackgroundPrecision genetic modifications: a new era in molecular biology and crop improvement
Fichtner, F., Urrea-Castellanos, R., & Ülker, B. (2014) · Planta · doi:10.1007/s00425-014-2029-y · Cited by 67
Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will
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“Alternatively, repurposing the gRNA as a scaffold to recruit activators via MS2-targeting has been proven effective: The authors fused several RNA hairpins from the male-specific bacteriophage-2 (MS2) to the 30end of a sgRNA and fused the MS2 coat protein (MCP), which binds the MS2 hairpin, to VP64, resulting in effic…”
Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression
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Off‐target assessment of CRISPR ‐ C as9 guiding RNA s in human i PS and mouse ES cells
Tan, E., Li, Y., Velasco‐Herrera, M. D. C., Yusa, K., & Bradley, A. (2014) · genesis · doi:10.1002/dvg.22835 · Cited by 66
“The target gRNA expression constructs were ordered as individual 455-bp gBlocks from IDT and cloned into the pCR-BluntII TOPO vector (Invitrogen) (Mali et al., 2013c).”
BackgroundMethodInfluentialCRISPR–Cas9-mediated genome editing and guide RNA design
Wiles, M. V., Qin, W., Cheng, A. W., & Wang, H. (2015) · Mammalian Genome · doi:10.1007/s00335-015-9565-z · Cited by 67
“When used as a genome editing tool in cultured cells, either electroporation or transfection is often used to deliver a plasmid containing a ubiquitous promoter driving Cas9 and sgRNA expression (Cong et al. 2013; Mali et al. 2013b).”
BackgroundMethodDeep mutational scanning of S. pyogenes Cas9 reveals important functional domains
Spencer, J. M., & Zhang, X. (2017) · Scientific Reports · doi:10.1038/s41598-017-17081-y · Cited by 65
Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
Höijer, I., Johansson, J., Gudmundsson, S., Chin, C.-S., Bunikis, I., Häggqvist, S., Emmanouilidou, A., Wilbe, M., den Hoed, M., Bondeson, M.-L., et al. (2020) · Genome biology · doi:10.1186/s13059-020-02206-w · Cited by 65
Efficacy and long-term safety of CRISPR/Cas9 genome editing in the SOD1-linked mouse models of ALS
Deng, H., Zhai, H., Shi, Y., Liu, G., Lowry, J., Liu, B., Ryan, É. B., Yan, J., Yang, Y., Zhang, N., et al. (2021) · Communications Biology · doi:10.1038/s42003-021-01942-4 · Cited by 66
CRISPR nuclease off-target activity and mitigation strategies
Wienert, B., & Cromer, M. K. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.1050507 · Cited by 66
Exploiting CRISPR / C as systems for biotechnology
Sampson, T. R., & Weiss, D. S. (2013) · BioEssays · doi:10.1002/bies.201300135 · Cited by 65
“dCas9 can be tethered to a transcriptional activator, such as the omega subunit of RNA polymerase within a bacterial system or VP64 in a mammalian system [16–19].”
BackgroundInfluentialBogerd, H. P., Kornepati, A. V. R., Marshall, J. B., Kennedy, E. M., & Cullen, B. R. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1516305112 · Cited by 62
CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential
Blenke, E. O., Evers, M. J. W., Mastrobattista, E., & John van der, O. O. S. T. (2016) · Journal of Controlled Release · doi:10.1016/j.jconrel.2016.08.002 · Cited by 63
Use of TALEs and TALEN Technology for Genetic Improvement of Plants
Khan, Z., Khan, S. H., Mubarik, M. S., Sadia, B., & Ahmad, A. (2016) · Plant Molecular Biology Reporter · doi:10.1007/s11105-016-0997-8 · Cited by 63
Brain Organoids as Tools for Modeling Human Neurodevelopmental Disorders
Adams, J. W., Cugola, F. R., & Muotri, A. R. (2019) · Physiology · doi:10.1152/physiol.00005.2019 · Cited by 68
CRISPR-Cas9: A Promising Genome Editing Therapeutic Tool for Alzheimer’s Disease—A Narrative Review
Barman, N. C., Khan, M. N. M., Islam, M., Nain, Z., Roy, R. K., Haque, A., & BARMAN, S. K. (2020) · Neurology and Therapy · doi:10.1007/s40120-020-00218-z · Cited by 65
On- and off-target effects of paired CRISPR-Cas nickase in primary human cells
Klermund, J., Rhiel, M., Köcher, T., Chmielewski, K. O., Bischof, J., Andrieux, G., el Gaz, M., Hainzl, S., Boerries, M., Cornu, T. I., et al. (2024) · Molecular Therapy · doi:10.1016/j.ymthe.2024.03.006 · Cited by 65
“” 32 Guiding two Cas9n RNPs to opposite strands in close proximity leads to a paired nick, causing a staggered DSB. 33,34 Ef fi cient DSB induction, evidenced by signi fi cant indel formation, occurs between the nicks if they are offset by up to 100 bp. 34 Consequently, off-target sites of the…”
Takeuchi, R., Choi, M. Y., & Stoddard, B. L. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1321030111 · Cited by 61
Staying on target with CRISPR-Cas
Carroll, D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2684 · Cited by 60
Endonucleases: new tools to edit the mouse genome
Wijshake, T., Baker, D. J., & van de Sluis, B. (2014) · Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease · doi:10.1016/j.bbadis.2014.04.020 · Cited by 61
Adenoviral vectors for in vivo delivery of CRISPR-Cas gene editors
Rice-Boucher, P. J., Cui, X., & Curiel, D. T. (2020) · Journal of Controlled Release · doi:10.1016/j.jconrel.2020.09.003 · Cited by 62
Ochiai, H., Miyamoto, T., Kanai, A., Hosoba, K., Sakuma, T., Kudo, Y., Asami, K., Ogawa, A., Watanabe, A., Kajii, T., et al. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1317008111 · Cited by 60
CRISPR/Cas9 and cancer targets: future possibilities and present challenges
White, M. K., & Khalili, K. (2016) · Oncotarget · doi:10.18632/oncotarget.7104 · Cited by 62
Expansion of CRISPR targeting sites in Bombyx mori
Zeng, B., Zhan, S., Wang, Y., Huang, Y., Xu, J. Q., Liu, Q., Li, Z., Huang, Y., & Tan, A. (2016) · Insect Biochemistry and Molecular Biology · doi:10.1016/j.ibmb.2016.03.006 · Cited by 60
Mammalian Synthetic Biology: Engineering Biological Systems
Black, J. B., Pérez‐Piñera, P., & Gersbach, C. A. (2017) · Annual Review of Biomedical Engineering · doi:10.1146/annurev-bioeng-071516-044649 · Cited by 60
Beyond editing to writing large genomes
Chari, R., & Church, G. M. (2017) · Nature Reviews Genetics · doi:10.1038/nrg.2017.59 · Cited by 60
“A single mutation to the Cas9 protein can generate a version that only creates a single-strand DNA cut (nick) 63,64 .”
BackgroundResultCRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge
Lee, C.-H. (2019) · Molecules · doi:10.3390/molecules24071349 · Cited by 64
Precision genome editing: A small revolution for glycobiology
Steentoft, C., Bennett, E., Schjoldager, K. T.-B. G., Vakhrushev, S. Y., Wandall, H. H., & Clausen, H. (2014) · Glycobiology · doi:10.1093/glycob/cwu046 · Cited by 62
“…form of FokI is a dimer, and ZFNs are therefore introduced in pairs with the target site consisting of two binding sequences between 9 and 18 bp in length, depending on the number of ZF domains, and a 5–7 bp spacer region in between (Händel et al. 2009; Shimizu et al. 2009; Wilson et al. 2013).”
BackgroundDevelopment of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells
Yoshioka, S., Fujii, W., Ogawa, T., Sugiura, K., & Naito, K. (2015) · Scientific Reports · doi:10.1038/srep18341 · Cited by 59
“The present mono-promoter-driven CRISPR/Cas9 system is expected to be effective for applications requiring the large-scale deletion of a target genome locus 3,7 or the offset-nicking method 23,24 , which need multiple gRNAs.”
Westphal, A., Cheng, W., Yu, J., Graßl, G. A., Krautkrämer, M., Holst, O., Föger, N., & Lee, K. (2016) · The Journal of Experimental Medicine · doi:10.1084/jem.20141461 · Cited by 59
“To minimize potential off-target effects, we applied a double-nicking strategy (Mali et al., 2013; Ran et al., 2013a), in which a pair of single-guide RNA (sgRNA)–Cas9nickase complexes was used to generate two adjacent DNA single-strand breaks (nicks) on opposite DNA strands within exon 49 of human…”
MethodTóth, E., Weinhardt, N., Bencsura, P., Huszár, K., Kulcsár, P. I., Tálas, A., Fodor, E., & Welker, E. (2016) · Biology Direct · doi:10.1186/s13062-016-0147-0 · Cited by 58
Rose, J. C., Popp, N. A., Richardson, C. D., Stephany, J. J., Mathieu, J., Wei, C. T., Corn, J. E., Maly, D. J., & Fowler, D. M. (2020) · Nature Communications · doi:10.1038/s41467-020-16542-9 · Cited by 61
Wang, K., Escobar, M., Li, J., Mahata, B., Goell, J., Shah, S. T., Cluck, M., & Hilton, I. B. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac582 · Cited by 60
“In parallel, nuclease-null deactivated, ‘dCas’ proteins have been developed that retain the ability to precisely target genomic DNA without genetic disruption after targeting, and these dCas-based platforms can be used as versatile, RNA-guided DNA binding domains (5–7).”
Chen, L., Tang, L., Xiang, H., Jin, L., Li, Q., Dong, Y., Wang, W., & Zhang, G. (2014) · GigaScience · doi:10.1186/2047-217x-3-24 · Cited by 62
“Alternatively, inactive Cas9 fused to an activator protein, such as VP64, may increase the expression of ZFP42 and POU5F1 [41].”
BackgroundHu, J. H., Davis, K. M., & Liu, D. R. (2016) · Cell chemical biology · doi:10.1016/j.chembiol.2015.12.009 · Cited by 61
“…activators and repressors (Bikard et al., 2013; Cheng et al., 2013; Gilbert et al., 2013; Konermann et al., 2013; Maeder et al., 2013; Mali et al., 2013a; Perez-Pinera et al., 2013; Qi et al., 2013), epigenetic modulators (Hilton et al., 2015; Kearns et al., 2015), and…”
BackgroundInfluentialMarx, N., Grünwald‐Gruber, C., Bydlinski, N., Dhiman, H., Ly, N. N., Klanert, G., & Borth, N. (2018) · Biotechnology Journal · doi:10.1002/biot.201700217 · Cited by 57
Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout
Knopp, Y., Geis, F. K., Heckl, D., Horn, S., Neumann, T., Kuehle, J., Meyer, J., Fehse, B., Baum, C., Morgan, M. A., et al. (2018) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2018.09.006 · Cited by 63
Progresses, Challenges, and Prospects of Genome Editing in Soybean (Glycine max)
Xu, H., Zhang, L., Zhang, K., & Ran, Y. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.571138 · Cited by 60
“For CRISPR system, different sgRNAs structure (Mali et al., 2013) and specificity of Cas9 such as high-fidelity SpCas9 variants (Zhang et al., 2017) can affect the cleavage on target and off targets sites.”
BackgroundSrikanth, P., & Young‐Pearse, T. L. (2014) · Journal of Neurogenetics · doi:10.3109/01677063.2014.881358 · Cited by 61
CRISPR Genome Editing Made Easy Through the CHOPCHOP Website
Labun, K., Krause, M., Cleuren, Y. N. T., & Valen, E. (2021) · Current Protocols · doi:10.1002/cpz1.46 · Cited by 57
“The three most important parameters for this experiment are the choice of homology arms, the choice of nickase (D10A or N863A; Mali et al., 2013), and the optimal spacing of gRNAs (Ran et al., 2013).”
BackgroundInfluentialThe Role of Recombinant AAV in Precise Genome Editing
Bijlani, S., Pang, K. M., Sivanandam, V., Singh, A., & Chatterjee, S. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.799722 · Cited by 63
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
Regulation of Endogenous Human Gene Expression by Ligand-Inducible TALE Transcription Factors
Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., & Barbas, C. F. (2013) · ACS Synthetic Biology · doi:10.1021/sb400114p · Cited by 59
CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells
Kim, E. J., Kang, K. H., & Ju, J. H. (2017) · The Korean Journal of Internal Medicine · doi:10.3904/kjim.2016.198 · Cited by 58
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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