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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
Open accessAbstract
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
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“Moreover, in-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [12]
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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
Self-citationMultiplex 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
Self-citationRNA-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
Repurposing 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
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
RNA-programmed genome editing in human cells
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013) · eLife · doi:10.7554/elife.00471 · Cited by 2,098
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt135 · Cited by 1,566
Genetic engineering of human pluripotent cells using TALE nucleases
Hockemeyer, D. F., Wang, H., Kiani, S., Lai, C. S., Gao, Q., Cassady, J. P., Cost, G. J., Zhang, L., Santiago, Y., Miller, J. C., et al. (2011) · Nature Biotechnology · doi:10.1038/nbt.1927 · Cited by 1,161
Hockemeyer, D. F., Soldner, F., Beard, C., Gao, Q., Mitalipova, M. M., DeKelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Zeitler, B., et al. (2009) · Nature Biotechnology · doi:10.1038/nbt.1562 · Cited by 1,064
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Venken, K. J. T., Sarrion‐Perdigones, A., Vandeventer, P. J., Abel, N. S., Christiansen, A. E., & Hoffman, K. L. (2015) · Wiley Interdisciplinary Reviews Developmental Biology · doi:10.1002/wdev.214 · Cited by 43
Personalised genome editing – The future for corneal dystrophies
Moore, T., Christie, K. A., Marshall, J., & Nesbit, M. A. (2018) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2018.01.004 · Cited by 44
“Initial genome-wide analyses with CRISPR/Cas9 found that Cas9 had variable specificity and was capable of off-target cleavage at sequences with close homology to that of the target guide sequence (Cho et al., 2014; Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013).”
BackgroundResultInfluentialHigh-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
Bessen, J. L., Afeyan, L. K., Dančík, V., Koblan, L. W., Thompson, D. B., Leichner, C., Clemons, P. A., & Liu, D. R. (2019) · Nature Communications · doi:10.1038/s41467-019-09987-0 · Cited by 42
Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes
Zhang, L., Rube, H. T., Vakulskas, C. A., Behlke, M. A., Bussemaker, H. J., & Pufall, M. A. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa231 · Cited by 45
Regulating CRISPR/Cas9 Function through Conditional Guide RNA Control
Brown, W., Zhou, W., & Deiters, A. (2020) · ChemBioChem · doi:10.1002/cbic.202000423 · Cited by 42
Gene therapy for Fabry disease: Progress, challenges, and outlooks on gene-editing
Domm, J. M., Wootton, S. K., Medin, J. A., & West, M. L. (2021) · Molecular Genetics and Metabolism · doi:10.1016/j.ymgme.2021.07.006 · Cited by 45
Petri, K., Kim, D. Y., Sasaki, K. E., Canver, M. C., Wang, X., Shah, H., Lee, H., Horng, J. E., Clement, K., Iyer, S., et al. (2021) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2021.04.05.438458 · Cited by 44
Jamal, M., Khan, F. A., Lin, D., Habib, Z., Dai, J., & Cao, G. (2016) · Current Issues in Molecular Biology · doi:10.21775/cimb.020.001 · Cited by 41
“High rates of mutagenesis have been observed for off-target sites when using gRNA with only 30% GC content (Fu et al., 2013; Pattanayak et al., 2013).”
BackgroundMethodInfluentialCas9-Based Genome Editing in Drosophila
Housden, B. E., Lin, S., & Perrimon, N. (2014) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-801185-0.00019-2 · Cited by 41
“In mammalian systems, several reports suggest that off-target mutations may be a significant issue associated with the use of the CRISPR system (Fu et al., 2013; Hsu et al., 2013; Mali, Aach, et al., 2013; Pattanayak et al., 2013).”
BackgroundGUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases
Zhu, L. J., Lawrence, M., Gupta, A., Pagès, H., Küçükural, A., Garber, M. G., & Wolfe, S. A. (2017) · BMC Genomics · doi:10.1186/s12864-017-3746-y · Cited by 42
“Deep sequencing analysis of potential off-target sites [11 – 14, 17, 18] from populations of SpCas9-sgRNA treated cells revealed that the majority of mismatched sequences are not appreciably cleaved, but that a subset of these “ off-target ” sites are functional [11 – 14, 17, 18], where up to six…”
BackgroundTherapeutic genome editing in cardiovascular diseases
Nishiga, M., Qi, L. S., & Wu, J. C. (2020) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2020.02.003 · Cited by 42
GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos
Zuo, E., Sun, Y., Wu, W., Yuan, T., Ying, W., Sun, H., Yuan, L., Steinmetz, L. M., Li, Y., & Yang, H. (2020) · Nature Protocols · doi:10.1038/s41596-020-0361-1 · Cited by 43
The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD
Wang, J.-Z., Wu, P., Shi, Z.-M., Xu, Y.-L., & Liu, Z.-J. (2017) · Brain and Development · doi:10.1016/j.braindev.2017.03.024 · Cited by 41
“could be deleted by utilizing Cas9 and a number of welldesigned gRNAs [1,21,29,32,33].”
Novel Genome-Editing Tools to Model and Correct Primary Immunodeficiencies
de Bruin, L. M. O., Volpi, S., & Musunuru, K. (2015) · Frontiers in Immunology · doi:10.3389/fimmu.2015.00250 · Cited by 44
Applications of TALENs and CRISPR/Cas9 in Human Cells and Their Potentials for Gene Therapy
Niu, J., Zhang, B., & Chen, H. (2014) · Molecular Biotechnology · doi:10.1007/s12033-014-9771-z · Cited by 41
“Transfection conditions: Reducing the concentrations of gRNA and Cas9 nuclease to reduce their expression in cells [32, 41, 42, 45].”
BackgroundMethodNegative DNA supercoiling induces genome-wide Cas9 off-target activity
Newton, M. D., Losito, M., Smith, Q. M., Parnandi, N., Taylor, B. J., Akçakaya, P., Maresca, M., van Eijk, P., Reed, S. H., Boulton, S. J., et al. (2023) · Molecular Cell · doi:10.1016/j.molcel.2023.09.008 · Cited by 41
Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips
Rivera, J. A., Mao, G., Владимирович, С. А., Panfilov, M., Hou, Q., Lindell, M., Chanez, C., Ritort, F., Jínek, M., & Deindl, S. (2024) · Science · doi:10.1126/science.adn5371 · Cited by 40
CRISPR‐Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering
Kirchner, M., & Schneider, S. (2015) · Angewandte Chemie International Edition · doi:10.1002/anie.201504741 · Cited by 39
Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system
Zhou, Y., Liu, Y., Hussmann, D., Brøgger, P., Al-Saaidi, R. A., Tan, S., Lin, L., Petersen, T. S., Zhou, G. Q., Bross, P., et al. (2016) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-015-2128-3 · Cited by 41
Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53
Hyodo, T., Rahman, M. L., Karnan, S., Ito, T., Toyoda, A., Ota, A., Wahiduzzaman, M., Tsuzuki, S., Okada, Y., Hosokawa, Y., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2019.12.064 · Cited by 43
Kim, D., Kang, B., & Kim, J. (2021) · Nature Protocols · doi:10.1038/s41596-020-00453-6 · Cited by 44
Eslami-Mossallam, B., Klein, M., van der Smagt, C. P., van der Sanden, K., Jones, S. K., Hawkins, J. A., Finkelstein, I. J., & Depken, M. (2022) · Nature Communications · doi:10.1038/s41467-022-28994-2 · Cited by 40
Paving the way towards precise and safe CRISPR genome editing
Śledziński, P., Dabrowska, M., Nowaczyk, M., & Olejniczak, M. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107737 · Cited by 43
Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing
Bekalu, Z. E., Panting, M., Holme, I. B., & Brinch‐Pedersen, H. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms241511920 · Cited by 43
A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila
Jiang, X., Ren, X., Sun, J., Wang, X., Qiao, H., Xu, B., Liu, L., & Ni, J. (2015) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2015.02.007 · Cited by 39
Reprogramming homing endonuclease specificity through computational design and directed evolution
Thyme, S. B., Boissel, S., Quadri, S. A., Nolan, T., Baker, D. A., Park, R. U., Kusak, L., Ashworth, J. R., & Baker, D. A. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt1212 · Cited by 39
Yang, X., Chen, L., He, J., & Yu, W. (2017) · Plant Cell Reports · doi:10.1007/s00299-017-2172-6 · Cited by 39
“Off-target mutations were reported in both human cells and plants at sites that differ by as many as 6 nt from ontarget sites (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013; Jacobs et al. 2015; Zhang et al. 2014).”
Tycko, J., Barrera, L. A., Huston, N. C., Friedland, A. E., Wu, X., Gootenberg, J. S., Abudayyeh, O. O., Myer, V. E., Wilson, C. J., & Hsu, P. D. (2018) · Nature Communications · doi:10.1038/s41467-018-05391-2 · Cited by 38
Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency
Chung, C.-H., Allen, A. G., Sullivan, N. T., Atkins, A., Nonnemacher, M. R., Wigdahl, B. L., & Dampier, W. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.10.008 · Cited by 40
Erwood, S., Laselva, O., Bily, T. M. I., Brewer, R. A., Rutherford, A. H., Bear, C. E., & Ivakine, E. A. (2020) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2020.05.002 · Cited by 40
A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing
Yang, L. C., & Chen, J. (2020) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2020.06.003 · Cited by 38
Ullah, M., Xia, L., Xie, S., & Sun, S. (2020) · Biotechnology and Applied Biochemistry · doi:10.1002/bab.2077 · Cited by 42
Myoediting: Toward Prevention of Muscular Dystrophy by Therapeutic Genome Editing
Zhang, Y., Long, C., Bassel‐Duby, R., & Olson, E. N. (2018) · Physiological Reviews · doi:10.1152/physrev.00046.2017 · Cited by 42
“For example, INDELs caused by SpCas9 off-target cleavage can be detected at certain sites with up to five mismatches relative to the on-target site (112, 152, 308).”
BackgroundReprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing
Alves, E., Taifour, S., Dolcetti, R., Chee, J., Nowak, A. K., Gaudieri, S., & Blancafort, P. (2021) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2021.04.009 · Cited by 40
Goel, K., & Ploski, J. E. (2022) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2022.914430 · Cited by 41
“SpCas9 mediated genome editing is reasonably accurate, but a number of studies have demonstrated that Cas9 can bind and induce DSBs at sites that are not entirely complementary to the gRNA sequence (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013).”
BackgroundProbing RNA–protein networks: biochemistry meets genomics
Campbell, Z. T., & Wickens, M. (2015) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2015.01.003 · Cited by 38
Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice
Lee, A. Y.-F., & Lloyd, K. C. K. (2014) · FEBS Open Bio · doi:10.1016/j.fob.2014.06.007 · Cited by 37
“A potential drawback as a result of offtarget genetic mutations induced by promiscuous cutting by Cas9 nuclease can be spurious and unintended phenotypic consequences [19,20].”
BackgroundMusunuru, K. (2017) · Journal of the American College of Cardiology · doi:10.1016/j.jacc.2017.10.002 · Cited by 38
Wegner, M., Diehl, V., Bittl, V., de Bruyn, R., Wiechmann, S., Matthess, Y., Hebel, M., Hayes, M. G. B., Schaubeck, S., Benner, C. W., et al. (2019) · eLife · doi:10.7554/elife.42549 · Cited by 38
Diurnal Control of Blood Pressure Is Uncoupled From Sodium Excretion
Johnston, J. G., Speed, J. S., Becker, B., Kasztan, M., Soliman, R. H., Rhoads, M. K., Tao, B., Jin, C., Geurts, A. M., Hyndman, K. A., et al. (2020) · Hypertension · doi:10.1161/hypertensionaha.119.13908 · Cited by 39
CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering
Saifaldeen, M., Al-Ansari, D. E., Ramotar, D., & Aouida, M. (2020) · Cells · doi:10.3390/cells9112518 · Cited by 40
Xiao, H., Hu, J., Huang, C., Feng, W., Liu, Y., Kumblathan, T., Tao, J., Xu, J., Le, X. C., & Zhang, H. (2023) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2023.117000 · Cited by 39
“The PAM-proximal mismatches are less tolerable than the PAM distal mismatches for the sgRNA-guided target recognition of the Cas9 system [114, 115].”
BackgroundApplications of CRISPR-Cas9 mediated genome engineering
Yang, X. P. (2015) · Military Medical Research · doi:10.1186/s40779-015-0038-1 · Cited by 38
“Off-site targeting is largely affected by the number, position, distribution of mismatches and Cas9 concentration, and may be a major concern in CRISPR/Cas9-medicated genome engineering [15,19-22].”
Maxwell, C. S., Jacobsen, T. L., Marshall, R., Noireaux, V., & Beisel, C. L. (2018) · Methods · doi:10.1016/j.ymeth.2018.02.016 · Cited by 35
“Previously developed in vitro assays are based on cleaving PAM library plasmids within in vitro conditions followed by either a positive [35,36,38] or negative [16,37] screen for cleavage.”
Exploiting CRISPR–Cas9 technology to investigate individual histone modifications
Vasquez, J.-J., Wedel, C., Cosentino, R. O., & Siegel, T. N. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky517 · Cited by 37
“(∼3Gb), numerous studies have reported that Cas9 can induce off-target mutations, even at sequences that differ by 5 nt from the targeted site (11,39,40).”
BackgroundOn-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes
Korablev, A. N., Lukyanchikova, V., Serova, I. A., & Баттулин, Н. Р. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21103604 · Cited by 37
The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses
Lin, H., Li, G., Peng, X., Deng, A., Ye, L., Shi, L., Wang, T., & He, J. (2021) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2021.590989 · Cited by 41
Methods for targeted mutagenesis in zebrafish using TALENs
Hwang, W. Y., Peterson, R. T., & Yeh, J.-R. J. (2014) · Methods · doi:10.1016/j.ymeth.2014.04.009 · Cited by 37
The Landscape of Signaling Pathways and Proteasome Inhibitors Combinations in Multiple Myeloma
Paradžik, T., Bandini, C., Mereu, E., Labrador, M., Taìana, E., Amodio, N., Neri, A., & Piva, R. (2021) · Cancers · doi:10.3390/cancers13061235 · Cited by 36
“By contrast, CRISPR-Cas9 technology can lead to the induction of the DNA damage causing cell cycle stalling and cell death, exon skipping or alternative splicing, which may result in the translation of functional truncated protein, and genetic compensation, leading to a partial rescue phenotype [49,50].”
BackgroundCRISPR-M: Predicting sgRNA off-target effect using a multi-view deep learning network
Sun, J., Guo, J. H., & Liu, J. (2024) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1011972 · Cited by 39
Brandl, C., Ortiz, O., Röttig, B., Wefers, B., Wurst, W., & Kühn, R. (2014) · FEBS Open Bio · doi:10.1016/j.fob.2014.11.009 · Cited by 38
CRISPR ‐Cas9 technology and its application in haematological disorders
Zhang, H., & McCarty, N. (2016) · British Journal of Haematology · doi:10.1111/bjh.14297 · Cited by 37
“In 2013, Nature Biotechnology simultaneously published a ser- ies of letters regarding NHEJ-mediated off-target mismatches in human cells (Fu et al, 2013; Hsu et al, 2013; Mali et al, 2013b; Pattanayak et al, 2013).”
BackgroundGeneration of RUNX 3 knockout pigs using CRISPR /Cas9‐mediated gene targeting
Kang, J., Ryu, J., Cho, B., Lee, E., Yun, Y., Ahn, S., Lee, J., Ji, D., Lee, K., & Park, K. (2016) · Reproduction in Domestic Animals · doi:10.1111/rda.12775 · Cited by 35
Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1
Yang, M., Wei, H., Wang, Y., Deng, J., Tang, Y., Zhou, L., Guo, G. F., & Tong, A. (2017) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2017.05.009 · Cited by 38
“Although CRISPR-Cas9 systems are widely used for genome editing, the range of sequences Cas9 can recognize is constrained by the need for a specific PAM.”
InfluentialImpact of Genetic Variation on CRISPR-Cas Targeting
Canver, M. C., Joung, J. K., & Pinello, L. (2018) · The CRISPR Journal · doi:10.1089/crispr.2017.0016 · Cited by 36
CRISPR/Cas9‐Based Genome Editing and its Applications for Functional Genomic Analyses in Plants
Li, J., Li, Y., & Ma, L. (2019) · Small Methods · doi:10.1002/smtd.201800473 · Cited by 36
Precise and broad scope genome editing based on high-specificity Cas9 nickases
Wang, Q., Liu, J., Janssen, J. M., Le Bouteiller, M., Frock, R. L., & Gonçalves, M. A. F. V. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkaa1236 · Cited by 41
“In keeping with these data, the 10–12 nts most proximal to the PAM have been proposed to constitute a ‘seed region’ in which DNA-gRNA mismatches are particularly detrimental for RGN activity (1,9).”
BackgroundSharma, A. K., Nymark, M., Flo, S., Sparstad, T., Bones, A. M., & Winge, P. (2021) · Plant Biotechnology Journal · doi:10.1111/pbi.13582 · Cited by 36
“Several previous studies show that mismatches in the seed region (the 10–12 base pairs adjacent to the PAM) are critical and determine the specificity of Cas9 compared to the distal part of the sgRNA sequence (non-seed sequence) (Hsu et al., 2013; Kim et al., 2013; Pattanayak et al., 2013).”
BackgroundBaker, M. (2014) · Nature Biotechnology · doi:10.1038/nbt.2863 · Cited by 36
Liver‐targeted gene therapy: Approaches and challenges
Aravalli, R. N., Belcher, J. D., & Steer, C. J. (2015) · Liver Transplantation · doi:10.1002/lt.24122 · Cited by 37
Taylor, J., & Woodcock, S. A. (2015) · SLAS DISCOVERY · doi:10.1177/1087057115590069 · Cited by 35
“More handson approaches also have been developed to reduce CRISPR-Cas OTEs.”
BackgroundInfluentialCurrent status of genome editing in vector mosquitoes: A review
Reegan, A. D., Ceasar, S. A., Paulraj, M. G., Savarimuthu Ignacimuthu, S. J., & Al-Dhabi, N. A. (2016) · BioScience Trends · doi:10.5582/bst.2016.01180 · Cited by 37
Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells
Havlicek, S., Shen, Y., Alpagu, Y., Bruntraeger, M., Zufir, N. B. M., Phuah, Z. Y., Fu, Z., Dunn, N. R., & Stanton, L. W. (2017) · Molecular Therapy · doi:10.1016/j.ymthe.2016.11.007 · Cited by 33
“We measured RFN specificity by a commonly used approach that takes advantage of previously described gRNA target sites with identified, known off-target mutation sites.(10,14,15) We expressed these gRNAs targeting CLTA, EMX1, and VEGFA in combination with WT SpCas9 or as paired gRNAs with different RFN variants in HEK…”
MethodSanjurjo-Soriano, C., & Kalatzis, V. (2018) · Neural Plasticity · doi:10.1155/2018/5056279 · Cited by 38
Engineering Resistance Against Viruses in Field Crops Using CRISPRCas9
Hinge, V. R., Chavhan, R. L., Kale, S. P., Suprasanna, P., & Kadam, U. S. (2021) · Current Genomics · doi:10.2174/1389202922666210412102214 · Cited by 35
Strategies for High-Efficiency Mutation Using the CRISPR/Cas System
Feng, S., Wang, Z., Li, A., Xie, X., Liu, J., Li, S., Li, Y., Wang, B., Hu, L., Yang, L., et al. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.803252 · Cited by 40
“Thus, to select nuclear-active mutants, two surrogate reporters based on the NHEJ and singlestrand annealing (SSA) have been published (Pattanayak et al., 2013).”
BackgroundCRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering
Singh, A., Chakraborty, D., & Maiti, S. (2016) · Chemical Society Reviews · doi:10.1039/c6cs00197a · Cited by 35
Safety, Security, and Policy Considerations for Plant Genome Editing
Wolt, J. D. (2017) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2017.03.005 · Cited by 37
Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo”
Lareau, C. A., Clement, K., Hsu, J. Y., Pattanayak, V., Joung, J. K., Aryee, M. J., & Pinello, L. (2018) · Nature Methods · doi:10.1038/nmeth.4541 · Cited by 32
Merlin, J. P. J., & Abrahamse, H. (2024) · Biomedicine & Pharmacotherapy · doi:10.1016/j.biopha.2024.117516 · Cited by 35
Lentiviral Vectors in Cancer Immunotherapy
Oldham, R. A. A., Berinstein, E. M., & Medin, J. A. (2015) · Immunotherapy · doi:10.2217/imt.14.108 · Cited by 36
Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells
Holkers, M., Cathomen, T., & Gonçalves, M. A. F. V. (2014) · Methods · doi:10.1016/j.ymeth.2014.02.017 · Cited by 36
In Vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay
Liu, W., Yu, H., Zhou, X., & Xing, D. (2016) · Analytical Chemistry · doi:10.1021/acs.analchem.6b02338 · Cited by 34
Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox
Karvelis, T., Gasiūnas, G., & Šikšnys, V. (2017) · Current Opinion in Microbiology · doi:10.1016/j.mib.2017.05.009 · Cited by 33
Using Biotechnology-Led Approaches to Uplift Cereal and Food Legume Yields in Dryland Environments
Dwivedi, S. L., Siddique, K. H. M., Farooq, M., Thornton, P. K., & Ortiz, R. O. (2018) · Frontiers in Plant Science · doi:10.3389/fpls.2018.01249 · Cited by 33
Protein Engineering Strategies to Expand CRISPR-Cas9 Applications
Ribeiro, L. F., Ribeiro, L. F. C., Barreto, M. Q., & Ward, R. J. (2018) · International Journal of Genomics · doi:10.1155/2018/1652567 · Cited by 36
“Imperfect base pairing outside the seed region can be tolerated and results in off-target Cas9 activity [25].”
BackgroundLabel-Free CRISPR/Cas9 Assay for Site-Specific Nucleic Acid Detection
Hu, J., Jiang, M., Liu, R., & Lv, Y. (2019) · Analytical Chemistry · doi:10.1021/acs.analchem.9b02641 · Cited by 34
Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells
Tang, L., Yang, F., He, X., Xie, H., Liu, X., Fu, J., Xi, H., Lu, X., Liu, C., Song, Z., et al. (2019) · Cell Regeneration · doi:10.1016/j.cr.2019.08.002 · Cited by 34
Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy
Bandara, R. A., Chen, Z. R., & Hu, J. (2021) · Cell & Bioscience · doi:10.1186/s13578-021-00662-w · Cited by 35
“These strategies include optimizing sgRNA sequence and expression level [33, 34], modifying the Cas9 protein [35] or using a different Cas protein [36].”
MethodEmerging Gene Therapeutics for Epidermolysis Bullosa under Development
Bischof, J., Hierl, M., & Koller, U. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25042243 · Cited by 36
CRISPR/Cas technology: a revolutionary approach for genome engineering
Zhang, L., & Zhou, Q. (2014) · Science China Life Sciences · doi:10.1007/s11427-014-4670-x · Cited by 33
Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System
Bai, Y., He, L., Li, P., Xu, K., Shao, S., Ren, C., Liu, Z., Wei, Z., & Zhang, Z. (2016) · G3 Genes Genomes Genetics · doi:10.1534/g3.116.027706 · Cited by 33
“So, off-target mutations remain a major concern for CRISPR/Cas9 (Cho et al. 2014; Tsai et al. 2015; Fu et al. 2013; Pattanayak et al. 2013; Veres et al. 2014; Zhang et al. 2015) because it is likely to cause unwanted chromosomal rearrangements (Cho et al. 2014).”
BackgroundOvando-Roche, P., Georgiadis, A., Smith, A. J., Pearson, R. A., & Ali, R. R. (2017) · Current Stem Cell Reports · doi:10.1007/s40778-017-0078-4 · Cited by 35
Generation of complement protein C3 deficient pigs by CRISPR/Cas9-mediated gene targeting
Zhang, W., Wang, G., Wang, Y., Jin, Y., Zhao, L., Xiong, Q., Zhang, L., Mou, L., Li, R., Yang, H., et al. (2017) · Scientific Reports · doi:10.1038/s41598-017-05400-2 · Cited by 33
Henser‐Brownhill, T., Monserrat, J., & Scaffidi, P. (2017) · Epigenetics · doi:10.1080/15592294.2017.1395121 · Cited by 33
“plete loss of the target protein through gene knockout offers several advantages compared to RNA interferencebased methods, including improved signal-to-noise ratio and lower off-target effects [16],[17].”
BackgroundPrecise Cas9 targeting enables genomic mutation prevention
Chavez, A., Pruitt, B. W., Tuttle, M. L., Shapiro, R. S., Cecchi, R. J., Winston, J., Turczyk, B. M., Tung, M., Collins, J. J., & Church, G. M. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1718148115 · Cited by 32
Application of CRISPR genetic screens to investigate neurological diseases
So, R. W. L., Chung, S., Lau, H. H. C., Watts, J. J., Gaudette, E. V., Al‐Azzawi, Z. A. M., Bishay, J., Lin, L. T., Joung, J., Wang, X., et al. (2019) · Molecular Neurodegeneration · doi:10.1186/s13024-019-0343-3 · Cited by 37
A Small Molecule-Controlled Cas9 Repressible System
Wu, Y., Yang, L., Chang, T., KANDEEL, F. R., & Yee, J. (2020) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2019.12.026 · Cited by 32
Bennion, B. G., Croft, C. A., Ai, T. L., Qian, W. P., Menos, A. M., Miner, C. A., Frémond, M., Doisne, J., Andhey, P. S., Platt, D. J., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2020.107771 · Cited by 35
“Since CRISPR/Cas9 can sometimes produce offtarget effects (Cradick et al., 2013; Frock et al., 2015; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Wang et al., 2015), one potential question regarding the LN deficiency phenotype is whether a second mutation due to an off-target effect may be responsible f…”
BackgroundCRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells
Kimura, Y., Oda, M., Nakatani, T., Sekita, Y., Monfort, A., Wutz, A., Mochizuki, H., & Nakano, T. (2015) · Scientific Reports · doi:10.1038/srep10710 · Cited by 33
CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling
Torres-Ruiz, R., & Rodríguez, S. (2015) · International Journal of Molecular Sciences · doi:10.3390/ijms160922151 · Cited by 37
“The CRISPR-Cas9 system can induce off-target mutations at sites that are highly homologous to on-target sites [11,69–72].”
BackgroundDesign of efficacious somatic cell genome editing strategies for recessive and polygenic diseases
Carlson-Stevermer, J., Das, A., Abdeen, A. A., Fiflis, D., Grindel, B. I., Saxena, S., Akcan, T., Alam, T., Kletzien, H., Kohlenberg, L., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-20065-8 · Cited by 32
Gene editing for dyslipidemias: New tools to “cut” lipids
Stankov, S. G., & Cuchel, M. (2023) · Atherosclerosis · doi:10.1016/j.atherosclerosis.2023.01.010 · Cited by 34
CRISPR/Cas9: a powerful tool in colorectal cancer research
Hu, Y., Liu, L., Jiang, Q., Fang, W., Chen, Y., Hong, Y., & Zhai, X. (2023) · Journal of Experimental & Clinical Cancer Research · doi:10.1186/s13046-023-02901-z · Cited by 33
“One of the major off-target factors is the unreason-able gRNA design and selection, previous evidences have demonstrated that reasonable sgRNA can significantly reduce off-target rate and minimize unexpectable cleavages and their products [127].”
BackgroundAnti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering
Liu, Q., Zhang, H., & Huang, X. (2020) · The FEBS Journal · doi:10.1111/febs.15139 · Cited by 31
Synthetic chimeric nucleases function for efficient genome editing
Liu, R., Liang, L., Freed, E. F., Chang, H., Oh, E. J., Liu, Z., Garst, A. D., Eckert, C. A., & Gill, R. T. (2019) · Nature Communications · doi:10.1038/s41467-019-13500-y · Cited by 33
Folkerts, J., Gaudenzio, N., Maurer, M., Hendriks, R. W., Stadhouders, R., Tam, S., & Galli, S. J. (2020) · Nature Protocols · doi:10.1038/s41596-019-0288-6 · Cited by 34
CRISPR/Cas genome editing in grapevine: recent advances, challenges and future prospects
Ren, C., Lin, Y., & Liang, Z. (2022) · Fruit Research · doi:10.48130/frures-2022-0007 · Cited by 37
Double-strand break toxicity is chromatin context independent
Friskes, A., Koob, L., Krenning, L., Severson, T., Koeleman, E. S., Vergara, X., Schubert, M., van den Berg, J., Evers, B., Manjón, A. G., et al. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac758 · Cited by 32
“This could be explained by the fact that we use excessive amounts of Cas9/crRNA complexes which have been shown to tolerate mismatches in the guide matching region, ultimately leading to Cas9 editing (66).”
BackgroundGenome Editing for the Understanding and Treatment of Inherited Cardiomyopathies
Nguyen, Q., Lim, K. R. Q., & Yokota, T. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21030733 · Cited by 33
Metadata
- Authors
- Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A
- Liu, David R
- Venue
- Nature Biotechnology
- Published
- 2013-08-11
- Volume
- 31
- Issue
- 9
- Pages
- 839-843
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3782611
- OpenAlex
- W2149402050
- Cited by
- 1,487
- References
- 21
- Citations by year
- 2026: 29 · 2025: 62 · 2024: 81 · 2023: 72 · 2022: 113 · 2021: 136 · 2020: 144 · 2019: 109 · 2018: 127 · 2017: 145 · 2016: 149 · 2015: 135 · 2014: 152 · 2013: 28
- FWCI
- 69.33
- 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
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (R01GM073794, T32GM007753)
- 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, seed sequence, in vitro selection, genome editing, PAM sequence, HEK293T cells, human genome
- MeSH terms
- Bacterial Proteins, DNA, Endonucleases, Genetic Engineering, Humans, Streptococcus pyogenes, Genome, Sequence Analysis, DNA, Genomics, HEK293 Cells, RNA, Small Untranslated, High-Throughput Nucleotide Sequencing
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