The DOI matches its Crossref record; 30 of the 30 references it cites were found in a registry or an index.
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.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
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]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007) · Cell · doi:10.1016/j.cell.2007.11.019 · Cited by 20,221
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · cited 2×
“25μg of reporter library; or (2) 2μg TALE-TF plasmid and 0.”
BackgroundRNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
Self-citationRepurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937
Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1208507109 · Cited by 2,769
CRISPR RNA Maturation by Trans-Encoded Small RNA and Host Factor RNase III
Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., Eckert, M. R., Vogel, J., & Charpentier, E. (2011) · Nature · doi:10.1038/nature09886 · Cited by 2,563
Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors
Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., & Bonas, U. (2009) · Science · doi:10.1126/science.1178811 · Cited by 2,470
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429
Flagged first, then by citations
Cited by
Sorted by citations
Items 401–500 of 1,001
Gene Drive for Mosquito Control: Where Did It Come from and Where Are We Headed?
Macias, V. M., Ohm, J. R., & Rasgon, J. L. (2017) · International Journal of Environmental Research and Public Health · doi:10.3390/ijerph14091006 · Cited by 109
Developments in the Tools and Methodologies of Synthetic Biology
Kelwick, R. J. R., MacDonald, J. T., Webb, A. J., & Freemont, P. S. (2014) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2014.00060 · Cited by 106
“…systems is a major challenge with applications in encapsulating artificial organelles or protocells (Choi and Montemagno, 2005; Agapakis et al., 2012; Hammer and Kamat, 2012; Mali et al., 2013), the precise detection and delivery of payloads (Sukhorukov et al., 2005; Uchida et al., 2007).”
Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts
Howden, S. E., Maufort, J. P., Duffin, B. M., Elefanty, A. G., Stanley, E. G., & Thomson, J. A. (2015) · Stem Cell Reports · doi:10.1016/j.stemcr.2015.10.009 · Cited by 99
Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation
Braun, C. J., Bruno, P. M., Horlbeck, M. A., Gilbert, L. A., Weissman, J. S., & Hemann, M. T. (2016) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1600582113 · Cited by 104
Transforming plant biology and breeding with CRISPR /Cas9, Cas12 and Cas13
Schindele, P., Wolter, F., & Puchta, H. (2018) · FEBS Letters · doi:10.1002/1873-3468.13073 · Cited by 110
A neuroscientist’s guide to transgenic mice and other genetic tools
Navabpour, S., Kwapis, J. L., & Jarome, T. J. (2019) · Neuroscience & Biobehavioral Reviews · doi:10.1016/j.neubiorev.2019.12.013 · Cited by 108
Himeda, C. L., Jones, T. I., & Jones, P. L. (2015) · Molecular Therapy · doi:10.1038/mt.2015.200 · Cited by 102
Gene therapy for visual loss: Opportunities and concerns
Lee, J. H., Wang, J.-H., Chen, J., Li, F., Edwards, T. L., Hewitt, A. W., & Liu, G. (2018) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2018.08.003 · Cited by 103
Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9
Ren, J., & Zhao, Y. (2017) · Protein & Cell · doi:10.1007/s13238-017-0410-x · Cited by 103
“A pair of Cas9 nickases could generate two single-strand breaks adjacent to each other on opposite DNA strands when guided by two properly designed sgRNAs (Mali et al., 2013a; Ran et al., 2013; Cho et al., 2014).”
BackgroundGenome Editing Using Cas9 Nickases
Trevino, A. E., & Zhang, F. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00008-8 · Cited by 106
Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice
Mikami, M., Toki, S., & Endo, M. (2016) · Plant and Cell Physiology · doi:10.1093/pcp/pcw049 · Cited by 97
“…repeats)/Cas9 (CRISPR-associated protein 9) has been utilized widely as a genome editing tool in various organisms, including bacteria, yeast, animals and plants (Jinek et al. 2012, Cong et al. 2013, Cho et al. 2013, Hwang et al. 2013, Jiang et al. 2013a, Jinek et al. 2013, Mali et al. 2013a).”
InfluentialCRISPR/Cas System and Factors Affecting Its Precision and Efficiency
Javaid, N., & Choi, S. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.761709 · Cited by 106
CRISPR-Cas systems: new players in gene regulation and bacterial physiology
Sampson, T. R., & Weiss, D. S. (2014) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2014.00037 · Cited by 98
“There is precedence for this, at least in synthetically mutated Cas9 proteins (dCas9), which lack endonuclease activity, but retain the ability to bind DNA (Bikard et al., 2013; Cheng et al., 2013; Esvelt et al., 2013; Mali et al., 2013; Perez-Pinera et al., 2013; Qi et al., 2013).”
BackgroundMethodTargeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases
Standage-Beier, K., Zhang, Q., & Wang, X. (2015) · ACS Synthetic Biology · doi:10.1021/acssynbio.5b00132 · Cited by 96
Genome Editing in Human Stem Cells
Byrne, S. M., Mali, P., & Church, G. M. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00006-4 · Cited by 101
CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications
Cheng, H., Zhang, F., & Ding, Y. (2021) · Pharmaceutics · doi:10.3390/pharmaceutics13101649 · Cited by 100
Plant genome engineering in full bloom
Lozano‐Juste, J., & Cutler, S. R. (2014) · Trends in Plant Science · doi:10.1016/j.tplants.2014.02.014 · Cited by 95
CRISPR-mediated genome editing and human diseases
Cai, L., Fisher, A. L., Huang, H., & Xie, Z. (2016) · Genes & Diseases · doi:10.1016/j.gendis.2016.07.003 · Cited by 83
Tadić, V., Josipović, G., Zoldoš, V., & Vojta, A. (2019) · Methods · doi:10.1016/j.ymeth.2019.05.003 · Cited by 99
CRISPR screens for functional interrogation of immunity
Shi, H., Doench, J. G., & Chi, H. (2022) · Nature reviews. Immunology · doi:10.1038/s41577-022-00802-4 · Cited by 102
Hit and go CAS9 delivered through a lentiviral based self-limiting circuit
Petris, G., Casini, A., Montagna, C., Lorenzin, F., Prandi, D., Romanel, A., Zasso, J., Conti, L., Demichelis, F., & Cereseto, A. (2017) · Nature Communications · doi:10.1038/ncomms15334 · Cited by 98
Improved specificity of TALE-based genome editing using an expanded RVD repertoire
Miller, J. C., Zhang, L., Xia, D. F., Campo, J. J., Ankoudinova, I., Guschin, D. Y., Babiarz, J., Meng, X., Hinkley, S. J., Lam, S. C., et al. (2015) · Nature Methods · doi:10.1038/nmeth.3330 · Cited by 98
Cherok, E., Xu, S., Li, S., Das, S., Meltzer, W. A., Zalzman, M., Wang, C., & Karbowski, M. (2016) · Molecular Biology of the Cell · doi:10.1091/mbc.e16-04-0208 · Cited by 94
“The PCR products were incorporated into AflII-linearized gRNA cloning vector (www.addgene.org/41824/) using Gibson assembly as described (Mali et al., 2013).”
MethodEpigenome engineering: new technologies for precision medicine
Sgro, A., & Blancafort, P. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa1000 · Cited by 96
Laible, G., Wei, J., & Wagner, S. (2014) · Biotechnology Journal · doi:10.1002/biot.201400193 · Cited by 94
Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs
Ferry, Q. R. V., Lyutova, R., & Fulga, T. A. (2017) · Nature Communications · doi:10.1038/ncomms14633 · Cited by 93
Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering
Nuñez, J. K., Harrington, L. B., & Doudna, J. A. (2016) · ACS Chemical Biology · doi:10.1021/acschembio.5b01019 · Cited by 92
CRISPR/Cas systems: Delivery and application in gene therapy
Huang, J., Zhou, Y., Li, J., Lü, A., & Liang, C. (2022) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2022.942325 · Cited by 102
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox
Koonin, E. V., Gootenberg, J. S., & Abudayyeh, O. O. (2023) · Biochemistry · doi:10.1021/acs.biochem.3c00159 · Cited by 95
Carte, J. M., Christopher, R. T., Smith, J. T., Olson, S., Barrangou, R., Moineau, S., Glover, C. V. C., Graveley, B. R., Terns, R. M., & Terns, M. P. (2014) · Molecular Microbiology · doi:10.1111/mmi.12644 · Cited by 95
“…of the Csn Type II-Asystem, comprised of Csn1 (Cas9) and associated RNAs, was recently successfully adapted as a promising tool for genome editing and gene expression control in human cells as well as multiple model organisms (reviewed in Mali et al., 2013a; Pennisi, 2013; Terns and Terns, 2014).”
MethodA CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells
Zhu, Z., Verma, N., Gonzãlez, F., Shi, Z., & Huangfu, D. (2015) · Stem Cell Reports · doi:10.1016/j.stemcr.2015.04.016 · Cited by 91
“There have been concerns about potential off-target mutagenesis with the CRISPR/Cas system (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013).”
BackgroundIn trans paired nicking triggers seamless genome editing without double-stranded DNA cutting
Chen, X., Janssen, J. M., Liu, J., Maggio, I., Jong, A. E. J. ‘., Mikkers, H. M. M., & Gonçalves, M. A. F. V. (2017) · Nature Communications · doi:10.1038/s41467-017-00687-1 · Cited by 92
CRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 98
Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications
Didovyk, A., Borek, B., Tsimring, L., & Hasty, J. (2016) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2016.06.003 · Cited by 90
Genome editing in cardiovascular diseases
Strong, A., & Musunuru, K. (2016) · Nature Reviews Cardiology · doi:10.1038/nrcardio.2016.139 · Cited by 91
Cas9, Cpf1 and C2c1/2/3―What's next?
Nakade, S., Yamamoto, T., & Sakuma, T. (2017) · Bioengineered · doi:10.1080/21655979.2017.1282018 · Cited by 92
“The Cas9n variant has been used in double-nicking strategy.(29,30) The dCas9 variant has broad applications including FokI-dCas9(31,32) and specific purposes other than conventional genome editing.”
MethodEffective gene editing by high-fidelity base editor 2 in mouse zygotes
Liang, P., Sun, H., Sun, Y., Zhang, X., Xie, X., Zhang, J., Zhang, Z., Chen, Y., Ding, C., Xiong, Y., et al. (2017) · Protein & Cell · doi:10.1007/s13238-017-0418-2 · Cited by 91
CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives
Chan, Y.-T., Lu, Y., Wu, J., Zhang, C., Tan, H., Bian, Z., Wang, N., & Feng, Y. (2022) · Theranostics · doi:10.7150/thno.71144 · Cited by 93
“Utilizing the Cas9 mutant nickase version (Cas9n) to create nicking of both DNA strands separately by a pair of gRNAs, the genome-editing results in site-specific DSB [21].”
MethodInfluentialSystematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells
Stanton, B. C., Siciliano, V., Ghodasara, A., Wróblewska, L., Clancy, K., Trefzer, A. C., Chesnut, J. D., Weiss, R., & Voigt, C. A. (2014) · ACS Synthetic Biology · doi:10.1021/sb5002856 · Cited by 88
Mekler, V. M., Станиславович, М. Л., Semenova, E., Kuznedelov, K. D., & Severinov, K. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw138 · Cited by 93
Beyond Native Cas9: Manipulating Genomic Information and Function
Mitsunobu, H., Teramoto, J., Nishida, K., & Kondo, A. (2017) · Trends in biotechnology · doi:10.1016/j.tibtech.2017.06.004 · Cited by 87
Bioengineering Solutions for Manufacturing Challenges in CAR T Cells
Piscopo, N. J., Mueller, K. P., Das, A., Hematti, P., Murphy, W. L., Palecek, S. P., Capitini, C. M., & Saha, K. (2017) · Biotechnology Journal · doi:10.1002/biot.201700095 · Cited by 93
Puchta, H. (2015) · The Plant Journal · doi:10.1111/tpj.13100 · Cited by 87
Kim, J. (2016) · Nature Protocols · doi:10.1038/nprot.2016.104 · Cited by 89
Development and Application of Base Editors
Yang, B., Yang, L. C., & Chen, J. (2019) · The CRISPR Journal · doi:10.1089/crispr.2019.0001 · Cited by 87
“32,33 Surprisingly, nCas9 monomer was also found to induce unexpected C-to-T base substitutions at on-target sites.”
Guide RNA engineering for versatile Cas9 functionality
Nowak, C. M., Lawson, S., Zerez, M., & Bleris, L. G. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw908 · Cited by 83
A technological and regulatory outlook on CRISPR crop editing
Globus, R., & Qimron, U. (2017) · Journal of Cellular Biochemistry · doi:10.1002/jcb.26303 · Cited by 89
“Last, to drastically reduce the off-target activity, we can use higher fidelity Cas9 or other Cas9 orthologes with higher fidelity, or shorten the gRNA.(77,78,79,80,81)”
Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption
Gopalappa, R., Suresh, B., Ramakrishna, S., & Kim, S. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky222 · Cited by 86
CRISPR mediated somatic cell genome engineering in the chicken
Véron, N., Qu, Z., Kipen, P. A. S., Hirst, C. E., & Marcelle, C. (2015) · Developmental Biology · doi:10.1016/j.ydbio.2015.08.007 · Cited by 71
Hallmarks of cancer: The CRISPR generation
Moses, C., García‐Bloj, B., Harvey, A. R., & Blancafort, P. (2018) · European Journal of Cancer · doi:10.1016/j.ejca.2018.01.002 · Cited by 90
In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining
Geisinger, J. M., Turan, S., Hernandez, S., Spector, L. P., & Calos, M. P. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkv1542 · Cited by 86
Deep learning improves the ability of sgRNA off-target propensity prediction
Liu, Q., Cheng, X., Liu, G., Li, B., & Liu, X. (2020) · BMC Bioinformatics · doi:10.1186/s12859-020-3395-z · Cited by 88
The therapeutic application of CRISPR/Cas9 technologies for HIV
Saayman, S. M., Ali, S. A., Morris, K. V., & Weinberg, M. S. (2015) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.2015.1036736 · Cited by 91
“Activation can be enhanced by using multiple non-overlapping sgRNAs targeted to the same promoter, which in turn recruit multiple copies of the dCas9-VP64 fusion proteins [14-16,30].”
BackgroundIntegrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments
Canver, M. C., Haeussler, M., Bauer, D. E., Orkin, S. H., Sanjana, N. E., Shalem, O., Yuan, G., Zhang, F., Concordet, J., & Pinello, L. (2018) · Nature Protocols · doi:10.1038/nprot.2018.005 · Cited by 91
CRISPR/Cas9‐Directed Genome Editing of Cultured Cells
Yang, L., Yang, J. L., Byrne, S., Pan, J., & Church, G. M. (2014) · Current Protocols in Molecular Biology · doi:10.1002/0471142727.mb3101s107 · Cited by 82
CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects
Paul, J. W., & Qi, Y. (2016) · Plant Cell Reports · doi:10.1007/s00299-016-1985-z · Cited by 90
“When paired together by two distinct gRNAs targeting adjacent genomic sequences, two nickases can generate a complete DSB with sticky ends (Mali et al. 2013b; Ran et al. 2013) (Fig.”
Genome editing in crop improvement: Present scenario and future prospects
Miglani, G. S. (2017) · Journal of Crop Improvement · doi:10.1080/15427528.2017.1333192 · Cited by 86
“cognate DNA, is used to localize transcriptional activator or repressors to specific DNA sequences to control transcriptional activation and repression (Gilbert et al. 2013; Mali et al. 2013b).”
BackgroundWangensteen, K. J., Wang, Y. J., Dou, Z., Wang, A. W., Mosleh‐Shirazi, E., Horlbeck, M. A., Gilbert, L. A., Weissman, J. S., Berger, S. L., & Kaestner, K. H. (2017) · Hepatology · doi:10.1002/hep.29626 · Cited by 84
“CRISPR/Cas9 activation (CRISPRa) combines a catalytically dead, mutant Cas9 (dCas9) with transcriptional activators to enable gRNA-mediated, locus-specific gene activation(11-21).”
A bistable genetic switch based on designable DNA-binding domains
Lebar, T., Bezeljak, U., Golob, A., Jerala, M., Kadunc, L., Pirš, B., Stražar, M., Vučko, D., Zupančič, U., Benčina, M., et al. (2014) · Nature Communications · doi:10.1038/ncomms6007 · Cited by 83
RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?
Unniyampurath, U., Pilankatta, R., & Krishnan, M. N. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17030291 · Cited by 88
Wang, J., Jiang, X., Zhao, L., Zuo, S., Chen, X., Zhang, L., Lin, Z., Zhao, X., Qin, Y., Zhou, X., et al. (2019) · Acta Pharmaceutica Sinica B · doi:10.1016/j.apsb.2019.09.003 · Cited by 83
Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells
Tao, R. X., Wang, Y., Jiao, Y., Hu, Y. F., Li, L., Jiang, L., Zhou, L., Qu, J., Chen, Q., & Yao, S. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac506 · Cited by 86
Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing
Sinclair, F., Begum, A. A., Dai, C. C., Tóth, I., & Moyle, P. M. (2023) · Drug Delivery and Translational Research · doi:10.1007/s13346-023-01320-z · Cited by 91
Plant–pathogen interactions: toward development of next-generation disease-resistant plants
Nejat, N., Rookes, J. E., Mantri, N. L., & Cahill, D. M. (2016) · Critical Reviews in Biotechnology · doi:10.3109/07388551.2015.1134437 · Cited by 86
Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease
Doetschman, T., & Georgieva, T. G. (2017) · Circulation Research · doi:10.1161/circresaha.116.309727 · Cited by 91
Williams, R. M., Senanayake, U., Artibani, M., Taylor, G., Wells, D., Ahmed, A., & Sauka-Spengler, T. (2018) · Development · doi:10.1242/dev.160333 · Cited by 77
“Previously published work in cell culture systems suggests that dCas9-targeted gene activation requires use of multiple guide RNAs (Mali et al., 2013), whereas dCas9-KRAB- or dCas9-LSD1mediated enhancer repression can be achieved with a single guide (Kearns et al., 2015).”
BackgroundXiao (2017) · doi:10.24272/j.issn.2095-8137.2017.033 · Cited by 81
“Additionally, the development of CRISPR/Cas9 technology has made transgenesis more extensively applicable (Mali et al., 2013).”
Genetic and epigenetic control of gene expression by CRISPR–Cas systems
Lo, A., & Qi, L. (2017) · F1000Research · doi:10.12688/f1000research.11113.1 · Cited by 72
“While these simple fusion proteins are able to activate the expression of reporters or endogenous genes, the potency of these constructs in transcriptional activation is quite modest, about 2-fold to 5-fold on average when using a single sgRNA 63,67,69–71 .”
BackgroundTargeting Host Factors to Treat West Nile and Dengue Viral Infections
Krishnan, M. N., & García-Blanco, M. A. (2014) · Viruses · doi:10.3390/v6020683 · Cited by 83
Imaging Specific Genomic DNA in Living Cells
Chen, B., Guan, J., & Huang, B. (2016) · Annual Review of Biophysics · doi:10.1146/annurev-biophys-062215-010830 · Cited by 77
“In cultured mammalian cells, electroporation, Nucleofection or Lipofecamine-mediated transfection have been used to transiently express Cas9 and sgRNAs (30, 36, 43, 90), which is sufficient to induce efficient genome editing.”
BackgroundMethodInfluentialHeman-Ackah, S. M., Bassett, A. R., & Wood, M. J. A. (2016) · Scientific Reports · doi:10.1038/srep28420 · Cited by 77
CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomesPreprint
Aach, J., Mali, P., & Church, G. M. (2014) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/005074 · Cited by 80
A simple, flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system
Kim, H., Kim, S., Ryu, J., Choi, M. K., Kweon, J., Kang, B., Ahn, H., Bae, S., Kim, J., Kim, J., et al. (2016) · Journal of Integrative Plant Biology · doi:10.1111/jipb.12474 · Cited by 78
“Cas9 nickase, lacking one of two nuclease active sites via a single substitution (D10A or H840A), has been used to increase the specificity of genome editing (Mali et al. 2013; Ran et al. 2015b).”
MethodThe clinical potential of gene editing as a tool to engineer cell‐based therapeutics
Ashmore‐Harris, C., & Fruhwirth, G. O. (2020) · Clinical and Translational Medicine · doi:10.1186/s40169-020-0268-z · Cited by 79
Vriend, L. E. M., Prakash, R., Chen, C.-C., Vanoli, F., Cavallo, F., Zhang, Y., Jasin, M., & Krawczyk, P. M. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw179 · Cited by 77
A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection
Dukhovny, A., Lamkiewicz, K., Chen, Q., Fricke, M., Jabrane‐Ferrat, N., Marz, M., Jung, J. U., & Sklan, E. H. (2019) · Journal of Virology · doi:10.1128/jvi.00211-19 · Cited by 78
Newer Gene Editing Technologies toward HIV Gene Therapy
Manjunath, N., Yi, G., Dang, Y., & Shankar, P. (2013) · Viruses · doi:10.3390/v5112748 · Cited by 77
Next stop for the CRISPR revolution: RNA‐guided epigenetic regulators
Vora, S., Tuttle, M. L., Cheng, J. Y., & Church, G. M. (2016) · FEBS Journal · doi:10.1111/febs.13768 · Cited by 75
Advanced Nanotheranostics of CRISPR/Cas for Viral Hepatitis and Hepatocellular Carcinoma
Kong, H., Ju, E., Yi, K., Xu, W., Lao, Y., Cheng, D., Zhang, Q., Tao, Y., Li, M., & Ding, J. (2021) · Advanced Science · doi:10.1002/advs.202102051 · Cited by 77
Duda, K., Lonowski, L. A., Kofoed-Nielsen, M., Ibarra, A., Delay, C. M., Kang, Q., Yang, Z., Pruett‐Miller, S. M., Bennett, E., Wandall, H. H., et al. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku251 · Cited by 81
Applications of CRISPR Genome Engineering in Cell Biology
Wang, F., & Qi, L. S. (2016) · Trends in Cell Biology · doi:10.1016/j.tcb.2016.08.004 · Cited by 79
Development and application of CRISPR/Cas9 technologies in genomic editing
Zhang, C., Quan, R., & Wang, J. (2018) · Human Molecular Genetics · doi:10.1093/hmg/ddy120 · Cited by 80
Precise genome-editing in human diseases: mechanisms, strategies and applications
Zheng, Y., Li, Y., Zhou, K., Li, T., VanDusen, N. J., & Hua, Y. (2024) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-024-01750-2 · Cited by 91
Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta
Johnson, R. A., Gurevich, V., Hayut, S. F., Samach, A., & Levy, A. A. (2014) · Plant Molecular Biology · doi:10.1007/s11103-014-0266-x · Cited by 76
“While previous studies had found that their rice (Zhou et al. 2014) or plant codon-optimized (Xu et al. 2014) Cas9 variants out-performed their humanized counterpart , their hCas9 variant (Cong et al. 2013) was not the same as the one that we tested (Nekrasov et al. 2013; Mali et al. 2013b).”
BackgroundMethodInfluentialBisaria, N., Jarmoskaite, I., & Herschlag, D. (2017) · Cell Systems · doi:10.1016/j.cels.2016.12.010 · Cited by 74
“…in reducing off-target cleavage have been reported, most recently in the CRISPR-Cas9 field, by making changes in the nuclease or the RNA guide (Doench et al., 2016; Fu et al., 2014; Kleinstiver et al., 2016, 2015; Mali et al., 2013; Ran et al., 2013; Slaymaker et al., 2016; Tsai et al., 2014).”
Seruggia, D., & Montoliu, L. (2014) · Transgenic Research · doi:10.1007/s11248-014-9823-y · Cited by 79
“Last year, four independent groups nonetheless reported that the RNA-guiding process tolerates several mismatches between the RNA guide and the target DNA sequence (Fu et al. 2013; Mali et al. 2013; Pattanayak et al. 2013; Hsu et al. 2013), questioning the specificity of the CRISPR–Cas system.”
BackgroundDevelopment and Application of CRISPR/Cas in Microbial Biotechnology
Ding, W., Zhang, Y., & Shi, S. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.00711 · Cited by 81
Seruggia, D., Fernández, A., Cantero, M., Pelczar, P., & Montoliu, L. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv375 · Cited by 76
Cutting back malaria: CRISPR/Cas9 genome editing of Plasmodium
Lee, M., Lindner, S. E., Lopez-Rubio, J.-J., & Llinás, M. (2019) · Briefings in Functional Genomics · doi:10.1093/bfgp/elz012 · Cited by 78
“For example, the strong viral transactivators such as VP64 that are routinely employed to increase transcription in mammalian systems (‘CRISPR activation’, ‘CRISPRa’) [52] were not thought to be able to function effectively in Plasmodium parasites.”
BackgroundAtkins, A., Chung, C.-H., Allen, A. G., Dampier, W., Gurrola, T. E., Sariyer, I. K., Nonnemacher, M. R., & Wigdahl, B. L. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.673022 · Cited by 78
“While CRISPR/Cas9 is the most versatile of these systems it has also exhibited a propensity for off-target activity (Hockemeyer et al., 2011; Mussolino et al., 2011; Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013; Yang et al., 2013; Cho et al., 2014; Lin et al., 201…”
BackgroundEfficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus
Xu, K., Ren, C., Liu, Z., Zhang, T., Zhang, T., Li, D., Wang, L., Yan, Q., Guo, L., Shen, J., et al. (2014) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-014-1679-z · Cited by 78
“To improve the specificity, several strategies have been suggested (1) avoiding potential off-target sequences by considering the tolerance of mismatches and PAM mutations [11–14]; (2) titrating the dosage of Cas9 and sgRNA to minimize the off-target activity [11–13]; (3) utilizing sgRNA with shorter guide sequence [3…”
MethodRong, Z., Zhu, S., Xu, Y., & Fu, X. (2014) · Protein & Cell · doi:10.1007/s13238-014-0032-5 · Cited by 73
“…short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, has been developed for efficient gene targeting in cells of various species, including zebrafish (Hwang et al., 2013), mouse (Wang et al., 2013), monkey (Niu et al., 2014), and human (Cong et al., 2013; Mali et al., 2013b).”
BackgroundPerenthaler, E., Yousefi, S., Niggl, E., & Barakat, T. S. (2019) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2019.00352 · Cited by 75
“…include transcription activating domains such as multiple repeats of the herpes simplex VP16 activation domain (VP64) (Maeder et al., 2013; Mali et al., 2013), the nuclear factor-κB (NF-κB) trans-activating subunit activation domain (p65) and human heat-shock factor 1 (HSF1) (Konermann et…”
BackgroundTranscription Activator-like Effectors: A Toolkit for Synthetic Biology
Moore, R. L., Chandrahas, A. S., & Bleris, L. G. (2014) · ACS Synthetic Biology · doi:10.1021/sb400137b · Cited by 73
CRISPR-Cas9 technology: applications and human disease modelling
Torres-Ruiz, R., & Rodríguez, S. (2016) · Briefings in Functional Genomics · doi:10.1093/bfgp/elw025 · Cited by 77
de la Fuente‐Núñez, C., & Lu, T. K. (2016) · Integrative Biology · doi:10.1039/c6ib00140h · Cited by 75
Chen, S., Hou, C., Bi, H., Wang, Y., Xu, J. Q., Li, M., James, A. A., Huang, Y., & Tan, A. (2017) · Journal of Virology · doi:10.1128/jvi.02465-16 · Cited by 74
Zimmer, A. M., Pan, Y. K., Chandrapalan, T., Kwong, R. W. M., & Perry, S. F. (2019) · Journal of Experimental Biology · doi:10.1242/jeb.175737 · Cited by 77
Targeted genome engineering techniques in Drosophila
Beumer, K. J., & Carroll, D. (2014) · Methods · doi:10.1016/j.ymeth.2013.12.002 · Cited by 72
Synthetic epigenetics—towards intelligent control of epigenetic states and cell identity
Jurkowski, T. P., Ravichandran, M., & Stepper, P. (2015) · Clinical Epigenetics · doi:10.1186/s13148-015-0044-x · Cited by 73
“For targeting epigenetic modification, a catalytically inactive Cas9 variant has to be used, which still can recognize and bind the target sequence, but cannot cleave it [78-80].”
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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
