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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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Potential of Genome Editing to Improve Aquaculture Breeding and Production
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“…editing tool (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013b) and transcriptional regulator (Cheng et al., 2013; Gilbert et al., 2013; Konermann et al., 2013; Maeder et al., 2013b; Mali et al., 2013a; Perez-Pinera et al., 2013; Qi et al., 2013) of endogenous genes in mammalian cells.”
Loss-of-function genetic tools for animal models: cross-species and cross-platform differences
Housden, B. E., Muhar, M., Gemberling, M. P., Gersbach, C. A., Stainier, D. Y. R., Seydoux, G., Mohr, S. E., Zuber, J. A., & Perrimon, N. (2016) · Nature Reviews Genetics · doi:10.1038/nrg.2016.118 · Cited by 225
CRISPR/Cas9 advances engineering of microbial cell factories
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Advances in Industrial Biotechnology Using CRISPR-Cas Systems
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Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation
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Enabling plant synthetic biology through genome engineering
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A Thermostable Cas9 with Increased Lifetime in Human Plasma
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CRISPR/Cas9 and Genome Editing in Drosophila
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“Fusion of the crRNA and tracrRNA into a w100 nt synthetic single guide or chimeric RNA (sgRNA or chiRNA) has further simplified this system, which then only requires two components to be expressed (Dahlem et al., 2012; Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013b) (Fig.”
BackgroundInfluentialAssessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Tao, J., Bauer, D. E., & Chiarle, R. (2023) · Nature Communications · doi:10.1038/s41467-023-35886-6 · Cited by 232
CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming
Liu, Y., Chen, Y., Daley, T., Wang, F., Cao, W., Bhate, S. S., Lin, X., Still, C., Liu, H., Zhao, D., et al. (2018) · Cell stem cell · doi:10.1016/j.stem.2018.09.003 · Cited by 208
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
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“To minimize or avoid RGEN off-target effects, we and others have proposed various methods, which include dimeric Cas9 systems (paired Cas9 nickases [Mali et al. 2013a; Ran et al. 2013; Cho et al. 2014] and dCas9-FokI [Guilinger et al. 2014; Tsai et al. 2014]), delivery of RGEN ribonucleoproteins…”
BackgroundMethodCRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems
Jiang, W., & Marraffini, L. A. (2015) · Annual Review of Microbiology · doi:10.1146/annurev-micro-091014-104441 · Cited by 219
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Rapidly evolving homing CRISPR barcodes
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Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9
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“Since we and others have reported RNA-guided genome editing in human cells in January, 2013 (Cho et al., 2013a; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013b), the CRISPRCas9 system has been widely used in many labs all around the world to modify genomes in various organisms and cells.”
BackgroundMethodSynthetic Biology—The Synthesis of Biology
Ausländer, S., Ausländer, D., & Fussenegger, M. (2016) · Angewandte Chemie International Edition · doi:10.1002/anie.201609229 · Cited by 210
Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox
Razzaq, A., Saleem, F., Kanwal, M., Mustafa, G., Yousaf, S., Arshad, H. M. I., Hameed, M. K., Khan, M. S., & Joyia, F. A. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20164045 · Cited by 216
Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9
Anders, C., Bargsten, K., & Jinek, M. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.02.020 · Cited by 201
“Catalytically inactive variants of Cas9 have also been repurposed for RNAguided gene expression control and for cellular imaging applications (Chen et al., 2013; Gilbert et al., 2013; Konermann et al., 2015; Mali et al., 2013a).”
BackgroundCRISPR / dC as9 platforms in plants: strategies and applications beyond genome editing
Moradpour, M., & Abdulah, S. N. A. (2019) · Plant Biotechnology Journal · doi:10.1111/pbi.13232 · Cited by 205
Computational approaches for effective CRISPR guide RNA design and evaluation
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Abadi, S., Yan, W. X., Amar, D., & Mayrose, I. (2017) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1005807 · Cited by 208
Various Aspects of a Gene Editing System—CRISPR–Cas9
Janik, E., Niemcewicz, M., Ceremuga, M., Krzowski, Ł., Saluk, J., & Bijak, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21249604 · Cited by 223
Strategies for nonviral nanoparticle‐based delivery of CRISPR/Cas9 therapeutics
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A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification
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“…Cas9 nuclease is directed by guide RNAs (gRNAs) targeting 20 bp sequences adjacent to a 50-NRG-30 sequence motif, and the resultant cleavage has been used to edit the genome in several species (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013b).”
BackgroundResultA chemical-inducible CRISPR–Cas9 system for rapid control of genome editing
Liu, K. I., Ramli, M. N., Woo, A., Wang, Y., Zhao, T., Zhang, X., Yim, D., Chong, B. Y., Ali, G., Chua, M. Z. H., et al. (2016) · Nature Chemical Biology · doi:10.1038/nchembio.2179 · Cited by 199
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Gene editing and CRISPR in the clinic: current and future perspectives
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“activation domain of herpes simplex viral protein VP16 (193, 242, 252, 253) (Figure 6C).”
BackgroundCRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids
Hendriks, D. F. G., Clevers, H. C., & Artegiani, B. (2020) · Cell stem cell · doi:10.1016/j.stem.2020.10.014 · Cited by 202
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“The Cas9 nuclease can be further engineered into a nickase, which only cleaves a single strand of DNA and the same strategy on the other strand enhances the specificity of site recognition and consistently minimizes off-target effects [67, [70] [71] [72] [73] .”
BackgroundTAL effectors – pathogen strategies and plant resistance engineering
Boch, J., Bonas, U., & Lahaye, T. (2014) · New Phytologist · doi:10.1111/nph.13015 · Cited by 191
Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy
Gori, J. L., Hsu, P. D., Maeder, M. L., Shen, S., Welstead, G. G., & Bumcrot, D. A. (2015) · Human Gene Therapy · doi:10.1089/hum.2015.074 · Cited by 200
“63 67 Electroporation has also been successfully used to deliver RNA to fertilized mouse embryos in order to generate genetically modified rodents.”
InfluentialTALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
Mussolino, C., Alzubi, J., Fine, E. J., Morbitzer, R., Cradick, T. J., Lahaye, T., Bao, G., & Cathomen, T. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku305 · Cited by 194
Genome Editing in Plants: Exploration of Technological Advancements and Challenges
Vats, S., Kumawat, S., Kumar, V., Patil, G., Joshi, T., Sonah, H., Sharma, T. R., & Deshmukh, R. K. (2019) · Cells · doi:10.3390/cells8111386 · Cited by 195
“For example, PTG could be used with Cas9 nickase to improve targeting fidelity [115–117] or with dCas9 transcriptional activator or repressor to manipulate multiple gene expression [118,119].”
MethodSpatiotemporal control of CRISPR/Cas9 gene editing
Zhuo, C., Zhang, J., Lee, J., Jiao, J., Cheng, D., Liu, L., Kim, H., Tao, Y., & Li, M. (2021) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-021-00645-w · Cited by 196
“fused the activation domain p65 or VP64 to dCas9 to form the fusion protein dCas9-VP64 or dCas9-p65, respectively.(38,41) These fusion proteins and sgRNA targeting Gal4 upstream activation sequence (UAS) were simultaneously transfected into HEK-293T cells, which expressed GFP proteins.”
MethodChen, B., Hu, J., Almeida, R., Liu, H., Balakrishnan, S., Covill‐Cooke, C., Lim, W. A., & Huang, B. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkv1533 · Cited by 190
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Engineering of high-precision base editors for site-specific single nucleotide replacement
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CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective
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Off-target predictions in CRISPR-Cas9 gene editing using deep learning
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Biallelic genome modification in F 0 Xenopus tropicalis embryos using the CRISPR/Cas system
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“…genomic sites containing mismatches to the 50 portion of the gRNA (Cong et al., 2013; Cradick et al., in press; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013; Sapranauskas et al., 1967), with a minority of studies suggesting off-target mutagenesis might be…”
BackgroundMethodTALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis
Wu, H., Wang, Y., Zhang, Y., Yang, M., Lv, J., Liu, J., & Zhang, Y. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1421587112 · Cited by 185
Directing cellular information flow via CRISPR signal conductors
Liu, Y., Zhan, Y., Chen, Z., He, A., Li, J., Wu, H., Liu, L., Zhuang, C., Lin, J., Guo, X., et al. (2016) · Nature Methods · doi:10.1038/nmeth.3994 · Cited by 186
Development of CRISPR-Cas systems for genome editing and beyond
Zhang, F. (2019) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583519000052 · Cited by 188
“Concurrent with our study, a second report of gene editing using Cas9 was published (Mali et al., 2013b).”
BackgroundBalboa, D., Weltner, J., Eurola, S., Trokovic, R., Wartiovaara, K., & Otonkoski, T. (2015) · Stem Cell Reports · doi:10.1016/j.stemcr.2015.08.001 · Cited by 178
“CRISPRs originate from prokaryotic immune response systems and have been engineered to suit genome editing and transcriptional control purposes (Mali et al., 2013a).”
BackgroundResultInfluentialYoshimi, K., Kaneko, T., Voigt, B., & Mashimo, T. (2014) · Nature Communications · doi:10.1038/ncomms5240 · Cited by 178
“The bacterial CRISPR/Cas system has been shown to be an efficient gene-targeting technology in mammalian cells 18–20 and many organisms 21–25 , including mice 26,27 and rats 12,28,29 .”
InfluentialGeneration of muscular dystrophy model rats with a CRISPR/Cas system
Nakamura, K., Fujii, W., Tsuboi, M., Tanihata, J., Teramoto, N., Takeuchi, S., Naito, K., Yamanouchi, K., & Nishihara, M. (2014) · Scientific Reports · doi:10.1038/srep05635 · Cited by 188
CRISPR-Cas9D10A nickase-based genotypic and phenotypic screening to enhance genome editing
Chiang, T.-W. W., le Sage, C., Larrieu, D., Demir, M., & Jackson, S. P. (2016) · Scientific Reports · doi:10.1038/srep24356 · Cited by 171
Genetic Improvement of Wheat for Drought Tolerance: Progress, Challenges and Opportunities
Bapela, T., Shimelis, H., Tsilo, T. J., & Mathew, I. (2022) · Plants · doi:10.3390/plants11101331 · Cited by 211
CRISPR-Cas9-mediated homology-directed repair for precise gene editing
Liao, H., Wu, J., VanDusen, N. J., Li, Y., & Zheng, Y. (2024) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2024.102344 · Cited by 197
Evaluating and enhancing target specificity of gene-editing nucleases and deaminases
Kim, D., Luk, K., Wolfe, S. A., & Kim, J.-S. (2019) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-013118-111730 · Cited by 171
CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish
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Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design
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TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis
Ikmi, A., McKinney, S. A., Delventhal, K. M., & Gibson, M. C. (2014) · Nature Communications · doi:10.1038/ncomms6486 · Cited by 165
Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells
Byrne, S. M., Ortiz, L., Mali, P., Aach, J., & Church, G. M. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku1246 · Cited by 158
Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system
Shao, S., Zhang, W., Hu, H., Xue, B., Qin, J., Sun, C., Sun, Y., Wei, W., & Sun, Y. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw066 · Cited by 166
Genome engineering using the CRISPR/Cas system
Horii, T. (2014) · World Journal of Medical Genetics · doi:10.5496/wjmg.v4.i3.69 · Cited by 167
Patterns of CRISPR/Cas9 activity in plants, animals and microbes
Bortesi, L., Zhu, C., Zischewski, J., Pérez, L., Bassié, L., Nadi, R., Forni, G., Lade, S. B., Soto, E., Jin, X., et al. (2016) · Plant Biotechnology Journal · doi:10.1111/pbi.12634 · Cited by 167
Creating Class I MHC–Null Pigs Using Guide RNA and the Cas9 Endonuclease
Reyes, L. M., Estrada, J., Wang, Z., Blosser, R. J., Smith, R. F., Sidner, R. A., Paris, L. L., Blankenship, R. L., Ray, C. N., Miner, A. C., et al. (2014) · The Journal of Immunology · doi:10.4049/jimmunol.1402059 · Cited by 161
CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field
Schaeffer, S. M., & Nakata, P. A. (2015) · Plant Science · doi:10.1016/j.plantsci.2015.09.011 · Cited by 166
“Off-target editing can still occur in some instances; however, at a rate 50 to 1000-fo ld lower than the traditional CRISPR/Cas9 system [26,27,29].”
BackgroundMethodIn 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
Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9
Chen, Y., Cao, J., Xiong, M., Petersen, A. J., Dong, Y. W., Tao, Y., Huang, C. T.-L., Du, Z., & Zhang, S. (2015) · Cell stem cell · doi:10.1016/j.stem.2015.06.001 · Cited by 167
“…2011; Moscou and Bogdanove, 2009; Wood et al., 2011), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system (Cong et al., 2013; Garneau et al., 2010; Gasiunas et al., 2012; Jinek et al., 2012, 2013; Mali et al., 2013b; Sapranauskas et al., 2011).”
InfluentialA general strategy to construct small molecule biosensors in eukaryotes
Feng, J., Jester, B. W., Tinberg, C. E., Mandell, D. J., Antunes, M. S., Chari, R., Morey, K. J., Rios, X., Medford, J. I., Church, G. M., et al. (2015) · eLife · doi:10.7554/elife.10606 · Cited by 158
“The CRISPR/Cas9 system has proved to be an invaluable tool for genome editing (Mali et al., 2013; DiCarlo et al., 2013; Gratz et al., 2013; Hwang, 2013).”
InfluentialWang, X., Hu, L., Hao, J., Liao, L., Chiu, Y.-T., Shi, M., & Wang, Y. (2019) · Nature Cell Biology · doi:10.1038/s41556-019-0292-7 · Cited by 155
Chen, F., Ding, X. L., Feng, Y., Seebeck, T., Jiang, Y., & Davis, G. D. (2017) · Nature Communications · doi:10.1038/ncomms14958 · Cited by 156
Advances in Engineering the Fly Genome with the CRISPR-Cas System
Bier, E., Harrison, M. M., O’Connor-Giles, K. M., & Wildonger, J. (2018) · Genetics · doi:10.1534/genetics.117.1113 · Cited by 155
“Created to reduceoff-targetmutations, nickaseCas9has been used by itself and in pairs to introduce single- and DSBs, respectively, to edit the genome (Mali et al. 2013a; Ran et al. 2013).”
BackgroundMethodUnbiased investigation of specificities of prime editing systems in human cells
Kim, D. Y., Moon, S. B., Ko, J., Kim, Y.-S., & Kim, D. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa764 · Cited by 162
Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis
Castel, B., Tomlinson, L., Locci, F., Yang, Y., & Jones, J. D. G. (2019) · PLoS ONE · doi:10.1371/journal.pone.0204778 · Cited by 151
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
Precise Gene Deletion and Replacement Using the CRISPR/Cas9 System in Human Cells
Zheng, Q., Cai, X., Tan, M. H., Schaffert, S. A., Arnold, C. P., Gong, X., Chen, C., & Huang, S. (2014) · BioTechniques · doi:10.2144/000114196 · Cited by 154
Mouse Genome Editing Using the CRISPR/Cas System
Harms, D. W., Quadros, R. M., Seruggia, D., Ohtsuka, M., Takahashi, G., Montoliu, L., & Gurumurthy, C. B. (2014) · Current Protocols in Human Genetics · doi:10.1002/0471142905.hg1507s83 · Cited by 153
“…the Analysis of Human Disease 15.7.1 Supplement 83 This system has been redesigned for gene-editing purposes (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013b; Terns and Terns, 2014) by combining crRNA and tracrRNA into a chimeric unit called a single guide RNA (sgRNA), and by…”
BackgroundMethodInfluentialQuantifying on- and off-target genome editing
Hendel, A., Fine, E. J., Bao, G., & Porteus, M. H. (2015) · Trends in biotechnology · doi:10.1016/j.tibtech.2014.12.001 · Cited by 151
“Finally, standard RGENs create blunt breaks [21] but can be designed to create DSBs with either 3’ or 5’ overhangs when used in a paired “nickase” configuration [22, 23].”
MethodOptimization of Genome Engineering Approaches with the CRISPR/Cas9 System
Li, K., Wang, G., Andersen, T., Zhou, P., & Pu, W. T. (2014) · PLoS ONE · doi:10.1371/journal.pone.0105779 · Cited by 123
“It has been suggested that a ‘‘dual nick’’ strategy, in which two gRNAs direct the Cas9D10A single strand nickase activity to nearby sites to yield an effective double strand break, will be a effective means to minimize off-target mutagenesis [11,13], because the two gRNAs will rarely yield nearby nicks outside of the…”
BackgroundAntimicrobial peptides from fish: beyond the fight against pathogens
Valero, Y., Saraiva‐Fraga, M., Costas, B., & Guardiola, F. A. (2018) · Reviews in Aquaculture · doi:10.1111/raq.12314 · Cited by 155
Modrzejewski, D., Hartung, F., Lehnert, H., Sprink, T., Kohl, C., Keilwagen, J., & Wilhelm, R. (2020) · Frontiers in Plant Science · doi:10.3389/fpls.2020.574959 · Cited by 151
“These results are consistent with previous findings (e.g., Mali et al., 2013; Endo et al., 2015; Jiang et al., 2015).”
ResultTALENs—an indispensable tool in the era of CRISPR: a mini review
Bhardwaj, A., & Nain, V. (2021) · Journal of Genetic Engineering and Biotechnology · doi:10.1186/s43141-021-00225-z · Cited by 162
Xu, P., Tong, Y., Liu, X., Wang, T., Li, C., Wang, B.-Y., Lv, X., Huang, Y., & Liu, D. (2015) · Scientific Reports · doi:10.1038/srep12065 · Cited by 152
“Recently,Cas9 nickase was also reported to mediate higher homologous recombination efficiency due to the producing single-strand overhangs 35,38,39 .”
High-efficiency targeted editing of large viral genomes by RNA-guided nucleases
Bi, Y., Sun, L., Gao, D., Ding, C., Li, Z., Li, Y., Cun, W., & Li, Q. (2014) · PLoS Pathogens · doi:10.1371/journal.ppat.1004090 · Cited by 148
“and potentially tolerates up to five target mismatches in human cells [12,24,25,26,27], thereby promoting undesired off-target mutations.”
BackgroundCRISPR technologies for bacterial systems: Current achievements and future directions
Choi, K. R., & Lee, S. Y. (2016) · Biotechnology Advances · doi:10.1016/j.biotechadv.2016.08.002 · Cited by 151
Li, P., Estrada, J., Burlak, C., Montgomery, J. B., Butler, J. R., Miyashiro, R., Wang, Z., Paris, L. L., Blankenship, R. L., Downey, S. M., et al. (2014) · Xenotransplantation · doi:10.1111/xen.12131 · Cited by 146
Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors
Hu, J., Lei, Y., Wong, W.-K., Liu, S., Lee, K.-C., He, X., You, W., Zhou, R., Guo, J., Chen, X., et al. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku109 · Cited by 145
Lampe, G. D., King, R. T., Halpin-Healy, T. S., Klompe, S. E., Hogan, M. I., Vo, P. L. H., Tang, S., Chavez, A., & Sternberg, S. H. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01748-1 · Cited by 154
Wolter, F., Klemm, J., & Puchta, H. (2018) · The Plant Journal · doi:10.1111/tpj.13893 · Cited by 144
Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K., & Tsai, S. Q. (2015) · Human Gene Therapy · doi:10.1089/hum.2015.084 · Cited by 147
Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering
Lee, K., Mackley, V. A., Rao, A., Chong, A. T., DeWitt, M. A., Corn, J. E., & Murthy, N. (2017) · eLife · doi:10.7554/elife.25312 · Cited by 147
“…with specificity, and can result in precise genome editing via homology directed repair (HDR) if a donor DNA template is simultaneously delivered along with the CRISPR/Cas9 system (Jinek et al., 2012; Cong et al., 2013; Cho et al., 2013; Mali et al., 2013a; DeWitt et al., 2016; Long et al., 2014).”
BackgroundCRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi
Bendixen, L., Jensen, T. I., & Bak, R. O. (2023) · Molecular Therapy · doi:10.1016/j.ymthe.2023.03.024 · Cited by 150
Yeast as a cell factory: current state and perspectives
Kavšček, M., Stražar, M., Curk, T., Natter, K., & Petrovič, U. (2015) · Microbial Cell Factories · doi:10.1186/s12934-015-0281-x · Cited by 150
Maggio, I., Holkers, M., Liu, J., Janssen, J. M., Chen, X., & Gonçalves, M. A. F. V. (2014) · Scientific Reports · doi:10.1038/srep05105 · Cited by 139
Li, J., Shou, J., Guo, Y., Tang, Y., Wu, Y., Jia, Z., Zhai, Y., Chen, Z., Xu, Q., & Wu, Q. (2015) · Journal of Molecular Cell Biology · doi:10.1093/jmcb/mjv016 · Cited by 144
“Potential off-target sites of sgRNAs were predicted by searching the human genome sequences (Supplementary Table S3) (Mali et al., 2013c; Wang et al., 2013; Lin et al., 2014).”
BackgroundInfluentialThe chemistry of Cas9 and its CRISPR colleagues
Chen, J. S., & Doudna, J. A. (2017) · Nature Reviews Chemistry · doi:10.1038/s41570-017-0078 · Cited by 143
High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis
Thomas, H. R., Percival, S. M., Yoder, B. K., & Parant, J. M. (2014) · PLoS ONE · doi:10.1371/journal.pone.0114632 · Cited by 137
“Recent advances in genome editing through ZFNs (Zinc-finger nucleases) [8–11], TALENs (transcription-activator–like effector nucleases) [12–15], or CRISPR (clustered regularly interspaced short palindromic repeats) nucleases [16–25], has opened up opportunities in many organisms and cell lines to genome manipulation a…”
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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