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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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Savell, K. E., Bach, S. V., Zipperly, M. E., Revanna, J. S., Goska, N. A., Tuscher, J. J., Duke, C. G., Sultan, F. A., Burke, J. N., Williams, D., et al. (2019) · eNeuro · doi:10.1523/eneuro.0495-18.2019 · Cited by 137
In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila
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Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing
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“Previous reports have documented that wild-type SpCas9 is able to induce DSBs at non-canonical PAMs at a lower frequency [46, 53].”
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Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System
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Genome editing at the crossroads of delivery, specificity, and fidelity
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Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex
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Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos
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CRISPR/Cas9 mediated genome engineering in Drosophila
Bassett, A. R., & Liu, J. (2014) · Methods · doi:10.1016/j.ymeth.2014.02.019 · Cited by 131
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Kulcsár, P. I., Tálas, A., Huszár, K., Ligeti, Z., Tóth, E., Weinhardt, N., Fodor, E., & Welker, E. (2017) · Genome biology · doi:10.1186/s13059-017-1318-8 · Cited by 134
Recent Advances in Genome Editing Using CRISPR/Cas9
Ding, Y., Li, H., Chen, L., & Xie, K. (2016) · Frontiers in Plant Science · doi:10.3389/fpls.2016.00703 · Cited by 144
“In the prototype of the CRISPR/Cas9 system, Cas9 was directed to the DNA target by an RNA duplex of crRNA and tracrRNA, but a single guide RNA (gRNA) is used instead for genome editing (Gasiunas et al., 2012; Jinek et al., 2012, 2013; Cong et al., 2013; Mali et al., 2013b).”
BackgroundMethodEş, İ., Gavahian, M., Martí-Quijal, F. J., Lorenzo, J. M., Khaneghah, A. M., Tsatsanis, C., Kampranis, S. C., & Barba, F. J. (2019) · Biotechnology Advances · doi:10.1016/j.biotechadv.2019.02.006 · Cited by 139
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Pamies, D. (2018) · ALTEX · doi:10.14573/altex.1710081 · Cited by 127
Genome-wide specificity of prime editors in plants
Jin, S., Lin, Q., Luo, Y., Zhu, Z., Liu, G., Li, Y., Chen, K., Qiu, J., & Gao, C. (2021) · Nature Biotechnology · doi:10.1038/s41587-021-00891-x · Cited by 131
Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology
Jusiak, B., Cleto, S., Pérez‐Piñera, P., & Lu, T. K. (2016) · Trends in biotechnology · doi:10.1016/j.tibtech.2015.12.014 · Cited by 132
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“2014), and/or reporter assays (Mali et al. 2013a) to inform the selection of off-target sites for direct interrogation.”
BackgroundMethodResultInfluentialMultiplex genome-editing technologies for revolutionizing plant biology and crop improvement
Abdelrahman, M., Wei, Z., Rohila, J. S., & Zhao, K. (2021) · Frontiers in Plant Science · doi:10.3389/fpls.2021.721203 · Cited by 125
“MGE tools have become more user-friendly, particularly after the development and adaptation of the CRISPR-Cas system (Jinek et al., 2012; Cho et al., 2013; Cong et al., 2013; Mali et al., 2013).”
MethodTu, Z., Yang, W., Yan, S., Guo, X., & Li, X. (2015) · Molecular Neurodegeneration · doi:10.1186/s13024-015-0031-x · Cited by 125
“Use of bioinformatic screening to search for unique genomic targets and use of paired Cas9 nickases can also reduce off-targets [59, 82].”
BackgroundProtospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage
Cencic, R., Miura, H., Malina, A., Robert, F., Ethier, S., Schmeing, T. M., Dostie, J., & Pelletier, J. (2014) · PLoS ONE · doi:10.1371/journal.pone.0109213 · Cited by 117
“Here, pairs of sgRNAs directed against a desired locus recruit Cas9 nickases (D10A or H840A mutants) to target the complementary DNA strands to within ,30 bp of each other [16,24,25].”
BackgroundEngineering cell signaling using tunable CRISPR–Cpf1-based transcription factors
Liu, Y., Han, J., Chen, Z., Wu, H., Dong, H., & Nie, G. (2017) · Nature Communications · doi:10.1038/s41467-017-02265-x · Cited by 126
BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity
Jiang, W., Feng, S., Huang, S., Yu, W., Li, G., Yang, G., Liu, Y., Zhang, Y., Zhang, L., Hou, Y., et al. (2018) · Cell Research · doi:10.1038/s41422-018-0052-4 · Cited by 120
CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences
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Precision Control of CRISPR-Cas9 Using Small Molecules and Light
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Advanced CRISPR/Cas-based genome editing tools for microbial biofuels production: A review
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A new age in functional genomics using CRISPR/Cas9 in arrayed library screening
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A simplified and efficient germline-specific CRISPR/Cas9 system forDrosophilagenomic engineering
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CRISPR/Cas9: A Tool to Circumscribe Cotton Leaf Curl Disease
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Genome Editing: A New Approach to Human Therapeutics
Porteus, M. H. (2015) · The Annual Review of Pharmacology and Toxicology · doi:10.1146/annurev-pharmtox-010814-124454 · Cited by 118
CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X
Karimova, M., Beschorner, N., Dammermann, W., Chemnitz, J., Indenbirken, D., Bockmann, J., Grundhoff, A. T., Lüth, S., Buchholz, F., Wiesch, J. S. Z., et al. (2015) · Scientific Reports · doi:10.1038/srep13734 · Cited by 116
Li, P., Kleinstiver, B. P., Leon, M., Prew, M. S., Navarro-Gomez, D., Greenwald, S. H., Pierce, E. A., Joung, J. K., & Liu, Q. (2018) · The CRISPR Journal · doi:10.1089/crispr.2017.0009 · Cited by 116
Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila
Gokcezade, J., Sienski, G., & Duchek, P. (2014) · G3 Genes Genomes Genetics · doi:10.1534/g3.114.014126 · Cited by 116
Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9
Bassett, A. R., Tibbit, C., Ponting, C. P., & Liu, J. (2013) · Biology Open · doi:10.1242/bio.20137120 · Cited by 116
“The authors would like to thank Dr Feng Zhang for plasmid pX330 (Addgene plasmid 42230 (Mali et al., 2013b)), Dr James Sutherland for plasmid pSTABLE1-Puro (González et al., 2011), Dr Haruhiko Siomi for the anti-AGO1 antibody (Miyoshi et al., 2005), the Drosophila Genomic Resource Centre (DGRC)…”
BackgroundMethodInfluentialDNA-binding-domain fusions enhance the targeting range and precision of Cas9
Bolukbasi, M. F., Gupta, A., Oikemus, S., Derr, A. G., Garber, M. G., Brodsky, M. H., Zhu, L. J., & Wolfe, S. A. (2015) · Nature Methods · doi:10.1038/nmeth.3624 · Cited by 117
Petersen, B., Frenzel, A., Lucas‐Hahn, A., Herrmann, D., Hassel, P., Klein, S., Ziegler, M., Hadeler, K., & Niemann, H. (2016) · Xenotransplantation · doi:10.1111/xen.12258 · Cited by 118
Park, J., Dempewolf, E., Zhang, W., & Wang, Z. (2017) · PLoS ONE · doi:10.1371/journal.pone.0179410 · Cited by 116
“After a single amino acid substitution of aspartic acid (D) to alanine (A) at the 10th amino acid in the RuvC-like domain, the Cas9 function is changed from endonuclease to nickase [1, 8].”
BackgroundZinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing
Waryah, C. B., Moses, C., Arooj, M., & Blancafort, P. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-7774-1_2 · Cited by 117
CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives
Lee, J. S., Grav, L. M., Lewis, N. E., & Faustrup Kildegaard, H. (2015) · Biotechnology Journal · doi:10.1002/biot.201500082 · Cited by 117
Precision cancer mouse models through genome editing with CRISPR-Cas9
Mou, H., Kennedy, Z., Anderson, D. G., Yin, H., & Xue, W. (2015) · Genome Medicine · doi:10.1186/s13073-015-0178-7 · Cited by 116
Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders
Wojtal, D., Kemaladewi, D. U., Malam, Z., Abdullah, S., Wong, T. W. Y., Hyatt, E., Baghestani, Z., Pereira, S., Stavropoulos, J., Mouly, V., et al. (2016) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2015.11.012 · Cited by 121
Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells
Vassena, R., Heindryckx, B., Peco, R., Pennings, G., Raya, Á., Sermon, K., & Veiga, A. (2016) · Human Reproduction Update · doi:10.1093/humupd/dmw005 · Cited by 117
“…has been predominantly used to generate mutations, as damaged DNA repaired through NHEJ occurs with higher mutagenicity (Cong et al., 2013; Mali et al., 2013b), while CRISPR/Cas9 single mutants (CRISPR/Cas9 nickases) are more suitable for gene targeting applications where a donor plasmid…”
BackgroundInfluentialEngineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system
Kipniss, N. H., Dingal, P. C. D. P., Abbott, T. R., Gao, Y., Wang, H., Dominguez, A. A., Labanieh, L., & Qi, L. S. (2017) · Nature Communications · doi:10.1038/s41467-017-02075-1 · Cited by 113
“Substituting equations (12), (13), (14) into Equation (15) yields a steady-state formula for GFP as a function of CNO ligand and ARRB2-dCas9,”
BackgroundCurrent and future prospects for CRISPR‐based tools in bacteria
Luo, M. L., Leenay, R. T., & Beisel, C. L. (2015) · Biotechnology and Bioengineering · doi:10.1002/bit.25851 · Cited by 116
“On the other hand, the nicking Cas9 could be driving homologous recombination by accelerating strand invasion, similar to nickase activity in eukaryotes (Cong et al., 2013; Mali et al., 2013b).”
BackgroundInfluentialLonowski, L. A., Narimatsu, Y., Riaz, A., Delay, C. M., Yang, Z., Niola, F., Duda, K., Ober, E. A., Clausen, H., Wandall, H. H., et al. (2017) · Nature Protocols · doi:10.1038/nprot.2016.165 · Cited by 111
How specific is CRISPR/Cas9 really?
O’Geen, H., Yu, A. S., & Segal, D. J. (2015) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2015.10.001 · Cited by 108
“or high-throughput reporter screens [7] (Figure 2, top).”
Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery
Bolukbasi, M. F., Gupta, A., & Wolfe, S. A. (2015) · Nature Methods · doi:10.1038/nmeth.3684 · Cited by 112
Farasat, I., & Salis, H. M. (2016) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1004724 · Cited by 114
“Further, to predict how a guide RNA controls target specificity, we used deep sequencing data [3,33,37,41] to compile a position-dependent, nearest neighbor binding model that accounts for canonical and non-canonical PAM recognition sites, R-loop formation, and mismatches with DNA Table 1.”
MethodInfluentialPrecise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases
Dabrowska, M., Juzwa, W., Krzyżosiak, W. J., & Olejniczak, M. (2018) · Frontiers in Neuroscience · doi:10.3389/fnins.2018.00075 · Cited by 111
“The efficiency of paired Cas9 nickase editing depends on the activity of two sgRNAs and the length of the target sequence between the two sgRNAs (Mali et al., 2013; Ran et al., 2013).”
Hanewich-Hollatz, M. H., Chen, Z., Hochrein, L. M., Huang, J., & Pierce, N. A. (2019) · ACS Central Science · doi:10.1021/acscentsci.9b00340 · Cited by 111
Cell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins
Hoffmann, M. D., Aschenbrenner, S., Große, S., Rapti, K., Domenger, C., Fakhiri, J., Mastel, M., Börner, K., Eils, R., Grimm, D., et al. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz271 · Cited by 112
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Genetic approaches to hypothalamic-pituitary-adrenal axis regulation
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CRISPR-mediated Activation of Latent HIV-1 Expression
Limsirichai, P., Gaj, T., & Schaffer, D. V. (2015) · Molecular Therapy · doi:10.1038/mt.2015.213 · Cited by 112
Zhao, J., Song, Y., & Liu, D. (2019) · Journal of Hematology & Oncology · doi:10.1186/s13045-019-0705-x · Cited by 111
A glance at genome editing with CRISPR–Cas9 technology
Barman, A., Deb, B., & Chakraborty, S. (2019) · Current Genetics · doi:10.1007/s00294-019-01040-3 · Cited by 107
Metabolic engineering of plant secondary metabolites: prospects and its technological challenges
Devi, A. M., Devi, K. K., Pukhrambam, P. D., Devi, M. L., & Das, S. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1171154 · Cited by 128
“The use of “paired nickases” where two gRNAs and Cas9 nickases are used to generate adjacent offset nicks at the target site to improve specificity (Mali et al., 2013; Ran et al., 2013a; Ran et al., 2013b).”
BackgroundThe Bacterial Origins of the CRISPR Genome-Editing Revolution
Sontheimer, E. J., & Barrangou, R. (2015) · Human Gene Therapy · doi:10.1089/hum.2015.091 · Cited by 113
Mouse Genome Engineering via CRISPR-Cas9 for Study of Immune Function
Pelletier, S. W., Gingras, S., & Green, D. R. (2015) · Immunity · doi:10.1016/j.immuni.2015.01.004 · Cited by 108
“Although a single nucleotide mismatch does not affect Cas9 activity, two or more mismatches, depending on their location with respect to the PAM sequence, decrease Cas9 activity (Fu et al., 2013; Hsu et al., 2013; Lin et al., 2014; Mali et al., 2013a; Pattanayak et al., 2013).”
BackgroundMethodInfluentialCationic Polymer‐Mediated CRISPR/Cas9 Plasmid Delivery for Genome Editing
Zhang, Z., Wan, T. Y., Chen, Y., Chen, Y., Sun, H., Cao, T., Songyang, Z., Tang, G., Wu, C., Yuan, P., et al. (2018) · Macromolecular Rapid Communications · doi:10.1002/marc.201800068 · Cited by 109
Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants
Hajiahmadi, Z., Movahedi, A., Wei, H., Li, D., Orooji, Y., Ruan, H., & Zhuge, Q. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20153719 · Cited by 112
“The targeting specificity of Cas9 depends on the seed sequence and the presence of the PAM adjacent to the target sequence, but off-target mutations can occur in the genome with as many as three to five mismatches within the PAM-distal region of the target [8,9].”
BackgroundTools for efficient genome editing; ZFN, TALEN, and CRISPR
Shamshirgaran, Y., Liu, J., Sumer, H., Verma, P. J., & Taheri-Ghahfarokhi, A. (2022) · Methods in molecular biology · doi:10.1007/978-1-0716-2301-5_2 · Cited by 114
CRISPR-based technologies: prokaryotic defense weapons repurposed
Terns, R. M., & Terns, M. P. (2014) · Trends in Genetics · doi:10.1016/j.tig.2014.01.003 · Cited by 112
Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution
Gilles, A. F., & Averof, M. (2014) · EvoDevo · doi:10.1186/2041-9139-5-43 · Cited by 110
“The requirement that these nicks coincide drastically improves targeting specificity compared to that of single CRISPR nucleases [44,117,119,124].”
InfluentialGenome Editing Tools in Plants
Mohanta, T. K., Bashir, T., Hashem, A., Allah, E. F. A., & Bae, H. (2017) · Genes · doi:10.3390/genes8120399 · Cited by 109
Jubair, L., Fallaha, S., & McMillan, N. A. J. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.08.012 · Cited by 107
Cas9-Based Genome Editing in Xenopus tropicalis
Nakayama, T., Blitz, I. L., Fish, M. B., Odeleye, A. O., Manohar, S., Cho, K. W. Y., & Grainger, R. M. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00017-9 · Cited by 106
dCas9: A Versatile Tool for Epigenome Editing
Brocken, D. J. W., Tark‐Dame, M., & Dame, R. T. (2017) · Current Issues in Molecular Biology · doi:10.21775/cimb.026.015 · Cited by 113
“Cas9 has been very rapidly and widely adopted (Doudna and Charpentier, 2014) as tool for genome editing (Cho et al., 2013a; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013b).”
BackgroundMethodInfluentialProfiling single-guide RNA specificity reveals a mismatch sensitive core sequence
Zheng, T., Hou, Y., Zhang, P., Zhang, Z., Xu, Y. M., Zhang, L., Niu, L., Yang, Y. Q., Liang, D., Fan, Y., et al. (2017) · Scientific Reports · doi:10.1038/srep40638 · Cited by 105
Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing
Tang, Y., & Fu, Y. (2018) · Cell & Bioscience · doi:10.1186/s13578-018-0255-x · Cited by 121
Kotani, H., Taimatsu, K., Ohga, R., Ota, S., & Kawahara, A. (2015) · PLoS ONE · doi:10.1371/journal.pone.0128319 · Cited by 106
CRISPR/Cas9-Enabled Multiplex Genome Editing and Its Application
Minkenberg, B., Wheatley, M. S., & Yang, Y. (2017) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2017.05.003 · Cited by 107
Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates
Thurtle‐Schmidt, D. M., & Lo, T. (2018) · Biochemistry and Molecular Biology Education · doi:10.1002/bmb.21108 · Cited by 103
Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity
Nguyen, D., Miyaoka, Y., Gilbert, L. A., Mayerl, S. J., Lee, B. H., Weissman, J. S., Conklin, B. R., & Wells, J. A. (2016) · Nature Communications · doi:10.1038/ncomms12009 · Cited by 107
Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering
Guha, T. K., Wai, A., & Hausner, G. (2017) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2016.12.006 · Cited by 117
Progress in Genome Editing Technology and Its Application in Plants
Zhang, K., Raboanatahiry, N. H., Zhu, B., & Li, M. (2017) · Frontiers in Plant Science · doi:10.3389/fpls.2017.00177 · Cited by 105
“The current dominance of the CRISPR-Cas9 approach is the result of several landmark studies that were published at the beginning of 2013 (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013b).”
BackgroundKun, R. S., Gomes, A. C. D. S., Hildén, K., Salazar-Cerezo, S., Mäkelä, M., & de Vries, R. P. (2019) · Biotechnology Advances · doi:10.1016/j.biotechadv.2019.02.017 · Cited by 107
Modeling Disease In Vivo With CRISPR/Cas9
Dow, L. E. (2015) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2015.07.006 · Cited by 111
CRISPR/Cas9: an advanced tool for editing plant genomes
Samanta, M. K., Dey, A., & Gayen, S. (2016) · Transgenic Research · doi:10.1007/s11248-016-9953-5 · Cited by 110
“Fu et al. (2014) also demonstrate that combining truncated sgRNAs with the multiplex nicking strategy described above can reduce off-target mutagenesis even further.”
BackgroundMethodInfluentialGeneration of myostatin‐knockout chickens mediated by D10A‐Cas9 nickase
Kim, G., Lee, J. H., Song, S., Kim, S. W., Han, J. S., Shin, S. P., Park, B., & Park, T. S. (2020) · The FASEB Journal · doi:10.1096/fj.201903035r · Cited by 104
Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation
Moroz, E., Satyapertiwi, D., Ramel, M., Høgset, H., Sunyovszki, I., Liu, Z., Wojciechowski, J. P., Zhang, Y., Grigsby, C. L., Brito, L., et al. (2020) · ACS Central Science · doi:10.1021/acscentsci.9b01093 · Cited by 104
Enabling functional genomics with genome engineeringPreprint
Hilton, I. B., & Gersbach, C. A. (2015) · Genome Research · doi:10.1101/gr.190124.115 · Cited by 106
“…with this class of activation domains, both when these effectors are localized in high density at adjacent sequences (Maeder et al. 2013b,c; Mali et al. 2013a; Perez-Pinera et al. 2013a,b) and when combined in cis as multimolecular complexes (Cheng et al. 2013; Chakraborty et al. 2014; Gao…”
BackgroundInfluentialDesignable DNA-binding domains enable construction of logic circuits in mammalian cells
Gaber, R., Lebar, T., Majerle, A., Šter, B., Dobnikar, A., Benčina, M., & Jerala, R. (2014) · Nature Chemical Biology · doi:10.1038/nchembio.1433 · Cited by 103
Comprehensive analysis of the specificity of transcription activator-like effector nucleases
Juillerat, A., Dubois, G., Valton, J., Thomas, S., Stella, S., Maréchal, A., Langevin, S., Benomari, N., Bertonati, C., Silva, G. H., et al. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku155 · Cited by 93
CRISPR/Cas9 for Human Genome Engineering and Disease Research
Xiong, X., Chen, M., Lim, W. A., Zhao, D., & Qi, L. S. (2016) · Annual Review of Genomics and Human Genetics · doi:10.1146/annurev-genom-083115-022258 · Cited by 111
Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency
Zhang, J., Li, X., Neises, A., Chen, W., Hu, L., Ji, G., Yu, J.-Y., Xu, J., Yuan, W., Cheng, T., et al. (2016) · Scientific Reports · doi:10.1038/srep28566 · Cited by 104
“Several approaches have been taken to improve the specificity of Cas9-sgRNA, including a paired nicking strategy 24,29,31 and dimeric Cas9-based system 32,33 .”
Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive Pluripotency
Adachi, K., Kopp, W., Wu, G., Heising, S., Greber, B., Stehling, M., Araúzo-Bravo, M. J., Boerno, S. T., Timmermann, B., Vingron, M., et al. (2018) · Cell stem cell · doi:10.1016/j.stem.2018.05.020 · Cited by 104
Harnessing CRISPR-Cas9 immunity for genetic engineering
Charpentier, E., & Marraffini, L. A. (2014) · Current Opinion in Microbiology · doi:10.1016/j.mib.2014.07.001 · Cited by 107
CRISPR interference and priming varies with individual spacer sequences
Xue, C., Seetharam, A. S., Musharova, O. S., Severinov, K., Brouns, S. J. J., Severin, A. J., & Sashital, D. G. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv1259 · Cited by 102
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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