Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases
Open accessAbstract
RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.
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The specificity of Cas9 activity can be enhanced by extending or truncating sgRNAs.
“accumulative evidence showed that the specificity of Cas9 activity can be enhanced by extending or truncating sgRNAs”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cho et al., 2014
The Results section (‘Guide RNA structure’; page unavailable) reports that adding two guanine nucleotides to the 5′ end of sgRNAs improved discrimination of off-target sites in the tested guides. It does not show that truncating sgRNAs enhances specificity; the shorter GX19 guides were less discriminating in this comparison. (LLM, conf=0.86)
Experimental — accuracy not yet measuredApplying truncated sgRNAs to paired Cas9 nickases achieves even more reduction of undesired mutagenesis.
“Even more reduction of undesired mutagenesis is achieved when truncated sgRNAs are applied to paired Cas9 nickases”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cho et al., 2014
The Results section (‘Paired Cas9 nickases’; page not provided) reports fewer detectable off-target indels with paired nickases than with Cas9 nucleases. The ‘Guide RNA structure’ section examines adding nucleotides to sgRNAs, not truncating them. Our copy does not show that truncated sgRNAs were applied to paired nickases or produced a further reduction. (LLM, conf=0.86)
Experimental — accuracy not yet measuredExtra guanine nucleotides at the 5' end of sgRNAs may hinder the interaction between the Cas9/sgRNA complex and the DNA at off-target sites.
“may hinder the interaction between the Cas9/sgRNA complex and the DNA at the off-target sites”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cho et al., 2014
In Results > Guide RNA structure (page unavailable), adding two 5′ guanines improved discrimination of off-target sites, with indels barely detected at six of seven validated sites. The paper does not show that the guanines hinder Cas9/sgRNA binding to off-target DNA; it instead suggests possible effects on guide RNA stability, concentration, or secondary structure. (LLM, conf=0.82)
Experimental — accuracy not yet measuredExtra guanine nucleotides at the 5' end of sgRNAs are favored in T7-promoter driven transcription.
“These extra guanine nucleotides are favored in T7-promoter driven transcription”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cho et al., 2014
Both the structure and composition of the guide RNA can affect the frequency of off-target effects.
“First, both the structure and composition of the guide RNA can affect the frequency of off-target effects.50”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [50]
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Cites
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 · see citing works
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 · see citing works
RNA-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 · see citing works
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741 · see citing works
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013) · Cell · doi:10.1016/j.cell.2013.04.025 · Cited by 3,495 · see citing works
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
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 · see citing works
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 2,707 · see citing works
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 · see citing works
A TALE nuclease architecture for efficient genome editing
Miller, J. C., Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., et al. (2011) · Nature Biotechnology · doi:10.1038/nbt.1755 · Cited by 2,098 · see citing works
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 · see citing works
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2507 · Cited by 1,863 · see citing works
Self-citationRNA-guided genetic silencing systems in bacteria and archaea
Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012) · Nature · doi:10.1038/nature10886 · Cited by 1,851 · see citing works
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · cited 4× · see citing works
“In line with two recent reports (Mali et al. 2013a; Ran et al. 2013), the repair of two nicks that would produce 59 overhangs led to the formation of indels much more frequently than those producing 39 overhangs at three genomic loci (Fig.”
BackgroundResultInfluentialHighly efficient endogenous human gene correction using designed zinc-finger nucleases
Urnov, F. D., Miller, J. C., Lee, Y.-L., Beausejour, C. M., Rock, J. M., Augustus, S., Jamieson, A. C., Porteus, M. H., Gregory, P. D., & Holmes, M. C. (2005) · Nature · doi:10.1038/nature03556 · Cited by 1,642 · see citing works
Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R. (2013) · Nature Biotechnology · doi:10.1038/nbt.2673 · Cited by 1,487 · cited 1× · see citing works
“Recently, three groups reported that RGENs had off-target effects in human cells (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundFLASH assembly of TALENs for high-throughput genome editing
Reyon, D., Tsai, S. Q., Khayter, C., Foden, J. A., Sander, J. D., & Joung, J. K. (2012) · Nature Biotechnology · doi:10.1038/nbt.2170 · Cited by 1,171 · see citing works
An improved zinc-finger nuclease architecture for highly specific genome editing
Miller, J. C., Holmes, M. C., Wang, J., Guschin, D. Y., Lee, Y.-L., Rupniewski, I., Beausejour, C. M., Waite, A. J., Wang, N. S., Kim, K. A., et al. (2007) · Nature Biotechnology · doi:10.1038/nbt1319 · Cited by 1,051 · see citing works
Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease
Gratz, S. J., Cummings, A. M., Nguyen, J. N., Hamm, D. C., Donohue, L. K., Harrison, M. M., Wildonger, J., & O’Connor-Giles, K. M. (2013) · Genetics · doi:10.1534/genetics.113.152710 · Cited by 959 · see citing works
Enhancing Gene Targeting with Designed Zinc Finger Nucleases
Bibikova, M. V., Beumer, K. J., Trautman, J. K., & Carroll, D. (2003) · Science · doi:10.1126/science.1079512 · Cited by 824 · see citing works
Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos
Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J., & Xi, J. (2013) · Cell Research · doi:10.1038/cr.2013.45 · Cited by 819 · see citing works
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity
Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T., & Cathomen, T. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr597 · Cited by 714 · see citing works
Efficient TALEN-mediated gene knockout in livestock
Carlson, D. F., Tan, W. S., Lillico, S. G., Štveráková, D., Proudfoot, C., Christian, M. L., Voytas, D. F., Long, C. R., Whitelaw, B., & Fahrenkrug, S. C. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1211446109 · Cited by 580 · see citing works
Generation of gene-modified mice via Cas9/RNA-mediated gene targeting
Shen, B., Zhang, J., Wu, H., Wang, J., Ma, K., Li, Z., Zhang, X., Zhang, P., & Huang, X. (2013) · Cell Research · doi:10.1038/cr.2013.46 · Cited by 556 · see citing works
An unbiased genome-wide analysis of zinc-finger nuclease specificity
Gabriel, R., Lombardo, A., Arens, A., Miller, J. C., Genovese, P., Kaeppel, C., Nowrouzi, A., Bartholomae, C. C., Wang, J., Friedman, G., et al. (2011) · Nature Biotechnology · doi:10.1038/nbt.1948 · Cited by 526 · see citing works
Performance comparison of exome DNA sequencing technologies
Clark, M. J., Chen, R., Lam, H. Y. K., Karczewski, K. J., Chen, R., Euskirchen, G. M., Butte, A. J., & Snyder, M. P. (2011) · Nature Biotechnology · doi:10.1038/nbt.1975 · Cited by 509 · see citing works
Ding, Q., Regan, S. N., Xia, Y., Oostrom, L. A., Cowan, C. A., & Musunuru, K. (2013) · Cell stem cell · doi:10.1016/j.stem.2013.03.006 · Cited by 502 · see citing works
Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases
Szczepek, M., Brondani, V., Büchel, J., Serrano, L., Segal, D. J., & Cathomen, T. (2007) · Nature Biotechnology · doi:10.1038/nbt1317 · Cited by 501 · see citing works
Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly
Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., & Kim, J.-S. (2009) · Genome Research · doi:10.1101/gr.089417.108 · Cited by 466 · see citing works
Self-citationRevealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection
Pattanayak, V., Ramirez, C. L., Joung, J. K., & Liu, D. R. (2011) · Nature Methods · doi:10.1038/nmeth.1670 · Cited by 453 · see citing works
A library of TAL effector nucleases spanning the human genome
Kim, Y., Kweon, J., Kim, A., Chon, J. K., Yoo, J. Y., Kim, H. J., Kim, S., Lee, C., Jeong, E., Chung, E., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2517 · Cited by 371 · see citing works
Self-citationMoehle, E. A., Rock, J. M., Lee, Y.-L., Jouvenot, Y., DeKelver, R. C., Gregory, P. D., Urnov, F. D., & Holmes, M. C. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0611478104 · Cited by 371 · see citing works
Knockout mice created by TALEN-mediated gene targeting
Sung, Y. H., Baek, I., Kim, D. H., Jeon, J., Lee, J., Lee, K., Jeong, D., Kim, J., & Lee, H. (2013) · Nature Biotechnology · doi:10.1038/nbt.2477 · Cited by 361 · see citing works
Self-citationTargeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
Santiago, Y., Chan, E. M., Liu, P., Orlando, S., Zhang, L., Urnov, F. D., Holmes, M. C., Guschin, D. Y., Waite, A. J., Miller, J. C., et al. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0800940105 · Cited by 358 · see citing works
Targeted chromosomal deletions in human cells using zinc finger nucleasesPreprint
Lee, H. J., Kim, E., & Kim, J. (2009) · Genome Research · doi:10.1101/gr.099747.109 · Cited by 250 · see citing works
Self-citationCho, S. W., Lee, J., Carroll, D., Kim, J., & Lee, J. (2013) · Genetics · doi:10.1534/genetics.113.155853 · Cited by 248 · see citing works
Self-citationLive-cell dSTORM with SNAP-tag fusion proteins
Klein, T., Löschberger, A., Proppert, S., Wolter, S., van de Linde, S., & Sauer, M. (2010) · Nature Methods · doi:10.1038/nmeth0111-7b · Cited by 247 · see citing works
Chromosomal translocations induced at specified loci in human stem cells
Brunet, E., Simsek, D., Tomishima, M. J., DeKelver, R. C., Choi, V. M., Gregory, P. D., Urnov, F. D., Weinstock, D. M., & Jasin, M. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0902076106 · Cited by 206 · see citing works
Human zinc fingers as building blocks in the construction of artificial transcription factors
Bae, K., Kwon, Y. D., Shin, H.-C., Hwang, M., Ryu, E.-H., Park, K., Yang, H.-Y., Lee, D.-K., Lee, Y., Park, J.-W., et al. (2003) · Nature Biotechnology · doi:10.1038/nbt796 · Cited by 202 · see citing works
Self-citationOusterout, D. G., Pérez‐Piñera, P., Thakore, P. I., Kabadi, A. M., Brown, M. T., Qin, X., Fedrigo, O. D., Mouly, V., Tremblay, J. P., & Gersbach, C. A. (2013) · Molecular Therapy · doi:10.1038/mt.2013.111 · Cited by 181 · see citing works
Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects
Ramirez, C. L., Certo, M. T., Mussolino, C., Goodwin, M. J., Cradick, T. J., McCaffrey, A. P., Cathomen, T., Scharenberg, A. M., & Joung, J. K. (2012) · Nucleic Acids Research · doi:10.1093/nar/gks179 · Cited by 174 · see citing works
Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleasesPreprint
Lee, H. J., Kweon, J., Kim, E., Kim, S., & Kim, J. (2011) · Genome Research · doi:10.1101/gr.129635.111 · Cited by 165 · see citing works
Self-citationPrecision genome engineering with programmable DNA-nicking enzymesPreprint
Kim, E., Kim, S., Kim, D. H., Choi, B., Choi, I., & Kim, J. (2012) · Genome Research · doi:10.1101/gr.138792.112 · Cited by 137 · see citing works
Self-citationTargeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzymePreprint
Wang, J., Friedman, G., Doyon, Y., Wang, N. S., Li, C. J., Miller, J. C., Hua, K. L., Yan, J. J., Babiarz, J., Gregory, P. D., et al. (2012) · Genome Research · doi:10.1101/gr.122879.111 · Cited by 135 · see citing works
Detection of structural variants and indels within exome data
Karakoç, E., Alkan, C., O’Roak, B. J., Dennis, M. Y., Vives, L., Mark, K. M. K., Rieder, M. J., Nickerson, D. A., & Eichler, E. E. (2011) · Nature Methods · doi:10.1038/nmeth.1810 · Cited by 118 · see citing works
TALENs and ZFNs are associated with different mutation signatures
Kim, Y., Kweon, J., & Kim, J. (2013) · Nature Methods · doi:10.1038/nmeth.2364 · Cited by 97 · see citing works
Self-citationPreassembled zinc-finger arrays for rapid construction of ZFNs
Kim, S., Lee, M., Kim, H., Kang, M., & Kim, J. (2010) · Nature Methods · doi:10.1038/nmeth0111-7a · Cited by 86 · see citing works
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The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062 · see citing works
CRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153 · see citing works
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016) · Nature · doi:10.1038/nature16526 · Cited by 2,673 · see citing works
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339 · see citing works
Self-citationGUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268 · see citing works
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2808 · Cited by 1,993 · see citing works
“…nickases 8 ), in which two gRNAs targeted to adjacent sites on opposite DNA strands each recruit a Cas9 variant (Cas9-D10A) that nicks DNA instead of cutting both strands 7, 9, 10 , can reduce mutation frequencies at known off-target sites of single gRNA-guided Cas9 nuclease in human cells 7, 10 .”
BackgroundResultInfluentialHaeussler, M., Schönig, K., Eckert, H., Eschstruth, A., Mianné, J., Renaud, J.-B., Schneider-Maunoury, S., Shkumatava, A., Teboul, L., Kent, J., et al. (2016) · Genome biology · doi:10.1186/s13059-016-1012-2 · Cited by 1,756 · see citing works
Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014) · Genome Research · doi:10.1101/gr.171322.113 · Cited by 1,856 · see citing works
Self-citationRational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation
Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J., & Root, D. E. (2014) · Nature Biotechnology · doi:10.1038/nbt.3026 · Cited by 1,617 · see citing works
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H. (2015) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2015.37 · Cited by 1,273 · see citing works
DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins
Woo, J. W., Kim, J., Kwon, S. I., Corvalán, C., Cho, S. W., Kim, H., Kim, S.-G., Kim, S.-T., Choe, S., & Kim, J.-S. (2015) · Nature Biotechnology · doi:10.1038/nbt.3389 · Cited by 1,219 · see citing works
Self-citationDelivering CRISPR: a review of the challenges and approaches
Lino, C. A., Harper, J. C., Carney, J. P., & Timlin, J. A. (2018) · Drug Delivery · doi:10.1080/10717544.2018.1474964 · Cited by 1,185 · see citing works
Digenome-seq: Genome-wide profiling of CRISPR-cas9 off-target effects in human cells
Kim, D., Bae, S., Park, J., Kim, E., Kim, S., Yu, H. R., Hwang, J., Kim, J.-I., & Kim, J.-S. (2015) · Nature Methods · doi:10.1038/nmeth.3284 · Cited by 992 · see citing works
Self-citationGenome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
Wu, X., Scott, D. A., Kriz, A. J., Chiu, A. C., Hsu, P. D., Dadon, D. B., Cheng, A. W., Trevino, A. E., Konermann, S., Chen, S., et al. (2014) · Nature Biotechnology · doi:10.1038/nbt.2889 · Cited by 944 · see citing works
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing
Tsai, S. Q., Wyvekens, N., Khayter, C., Foden, J. A., Thapar, V., Reyon, D., Goodwin, M. J., Aryee, M. J., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2908 · Cited by 952 · see citing works
Correction of a pathogenic gene mutation in human embryos
Ma, H., Marti-Gutierrez, N., Park, S.-W., Wu, J., Lee, Y., Suzuki, K., Koski, A., Ji, D., Hayama, T., Ahmed, R., et al. (2017) · Nature · doi:10.1038/nature23305 · Cited by 969 · see citing works
Self-citationMultiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition
Ren, J., Liu, X., Fang, C., Jiang, S., June, C. H., & Zhao, Y. (2017) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-16-1300 · Cited by 942 · see citing works
Guilinger, J. P., Thompson, D. B., & Liu, D. R. (2014) · Nature Biotechnology · doi:10.1038/nbt.2909 · Cited by 836 · see citing works
Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
Kuscu, C., Arslan, S., Singh, R., Thorpe, J., & Adli, M. (2014) · Nature Biotechnology · doi:10.1038/nbt.2916 · Cited by 777 · see citing works
DeepCRISPR: Optimized CRISPR guide RNA design by deep learning
Chuai, G., Ma, H., Yan, J., Chen, M., Hong, N., Xue, D., Zhou, C., Zhu, C., Chen, K., Duan, B., et al. (2018) · Genome biology · doi:10.1186/s13059-018-1459-4 · Cited by 641 · see citing works
Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B. M., Vertino, P. M., Stewart, F. J., & Bao, G. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku402 · Cited by 634 · see citing works
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA
Ramakrishna, S., Kwaku Dad, A.-B., Beloor, J., Gopalappa, R., Lee, S.-K., & Kim, H. (2014) · Genome Research · doi:10.1101/gr.171264.113 · Cited by 655 · see citing works
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“In the sgRNA extending approach, two guanine nucleotides are usually added at the 5’ end of sgRNAs (termed 5’-GGX20) (Cho et al., 2014).”
BackgroundInfluentialWang, H.-X., Li, M., Lee, C. M., Chakraborty, S., Kim, H.-W., Bao, G., & Leong, K. W. (2017) · Chemical Reviews · doi:10.1021/acs.chemrev.6b00799 · Cited by 560 · see citing works
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Lee, J. K., Jeong, E., Lee, J., Jung, M., Shin, E., Kim, Y.-H., Lee, K., Jung, I., Kim, D., Kim, S., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-05477-x · Cited by 525 · see citing works
Self-citationEfficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9
Mandal, P. K., Ferreira, L. M. R., Collins, R., Meissner, T. B., Boutwell, C. L., Friesen, M., Vrbanac, V., Garrison, B. S., Stortchevoi, A., Bryder, D., et al. (2014) · Cell stem cell · doi:10.1016/j.stem.2014.10.004 · Cited by 475 · see citing works
Veres, A., Gosis, B. S., Ding, Q., Collins, R., Ragavendran, A., Brand, H., Erdin, S., Cowan, C. A., Talkowski, M. E., & Musunuru, K. (2014) · Cell Stem Cell · doi:10.1016/j.stem.2014.04.020 · Cited by 474 · see citing works
Wang, X., Wang, Y., Wu, X., Wang, J., Wang, Y., Qiu, Z., Chang, T., Huang, H., Lin, R.-J., & Yee, J.-K. (2015) · Nature Biotechnology · doi:10.1038/nbt.3127 · Cited by 442 · see citing works
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457 · see citing works
The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo
Lin, S.-R., Yang, H.-C., Kuo, Y.-T., Liu, C.-J., Yang, T.-Y., Sung, K.-C., Lin, Y.-Y., Wang, H.-Y., Wang, C.-C., Shen, Y.-C., et al. (2014) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2014.38 · Cited by 386 · see citing works
Billon, P., Bryant, E. E., Joseph, S. A., Nambiar, T. S., Hayward, S. B., Rothstein, R., & Ciccia, A. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.08.008 · Cited by 363 · see citing works
Zischewski, J., Fischer, R., & Bortesi, L. (2017) · Biotechnology Advances · doi:10.1016/j.biotechadv.2016.12.003 · Cited by 323 · see citing works
A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing
Edraki, A., Mir, A., Ibraheim, R., Gainetdinov, I., Yoon, Y., Song, C.-Q., Cao, Y., Gallant, J., Xue, W., Rivera-Pérez, J. A., et al. (2019) · Molecular Cell · doi:10.1016/j.molcel.2018.12.003 · Cited by 265 · see citing works
Advances in therapeutic CRISPR/Cas9 genome editing
Savić, N., & Schwank, G. (2016) · Translational Research · doi:10.1016/j.trsl.2015.09.008 · Cited by 257 · see citing works
Subburaj, S., Chung, S. J., Lee, C., Ryu, S.-M., Kim, D. H., Kim, J.-S., Bae, S., & Lee, G.-J. (2016) · Plant Cell Reports · doi:10.1007/s00299-016-1937-7 · Cited by 237 · see citing works
Self-citationGenetically engineered pigs as models for human disease
Perleberg, C., Kind, A., & Schnieke, A. (2018) · Disease Models & Mechanisms · doi:10.1242/dmm.030783 · Cited by 222 · see citing works
Cas9-chromatin binding information enables more accurate CRISPR off-target prediction
Singh, R., Kuscu, C., Quinlan, A., Qi, Y., & Adli, M. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv575 · Cited by 217 · see citing works
A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
O'Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., & Segal, D. J. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv137 · Cited by 219 · see citing works
CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells
Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., Young, C. S., Mojadidi, M., Fitz-Gibbon, S., Cooper, A. R., et al. (2016) · Molecular Therapy · doi:10.1038/mt.2016.148 · Cited by 220 · see citing works
CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank
Geurts, M. H., de Poel, E., Amatngalim, G. D., Oka, R., Meijers, F. M., Kruisselbrink, E., van Mourik, P., Berkers, G., de Winter-de Groot, K. M., Michel, S., et al. (2020) · Cell Stem Cell · doi:10.1016/j.stem.2020.01.019 · Cited by 222 · see citing works
Biological plasticity rescues target activity in CRISPR knock outs
Smits, A. H., Ziebell, F., Joberty, G., Zinn, N., Mueller, W. F., Clauder-Münster, S., Eberhard, D., Fälth Savitski, M., Grandi, P., Jakob, P., et al. (2019) · Nature Methods · doi:10.1038/s41592-019-0614-5 · Cited by 219 · see citing works
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
Kim, D., Kim, S., Kim, S., Park, J., & Kim, J.-S. (2016) · Genome Research · doi:10.1101/gr.199588.115 · Cited by 208 · see citing works
Self-citationCRISPR-Cas9: a new and promising player in gene therapy
Xiao-Jie, L., Hui-Ying, X., Zun-Ping, K., Jin-Lian, C., & Li-Juan, J. (2015) · Journal of Medical Genetics · doi:10.1136/jmedgenet-2014-102968 · Cited by 200 · see citing works
Web-based design and analysis tools for CRISPR base editing
Hwang, G.-H., Park, J., Lim, K., Kim, S., Yu, J., Yu, E., Kim, S.-T., Eils, R., Kim, J.-S., & Bae, S. (2018) · BMC Bioinformatics · doi:10.1186/s12859-018-2585-4 · Cited by 198 · see citing works
Self-citationOff-target predictions in CRISPR-Cas9 gene editing using deep learning
Lin, J., & Wong, K.-C. (2018) · Bioinformatics · doi:10.1093/bioinformatics/bty554 · Cited by 185 · see citing works
Evaluating and enhancing target specificity of gene-editing nucleases and deaminases
Kim, D., Luk, K., Wolfe, S. A., & Kim, J.-S. (2019) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-013118-111730 · Cited by 171 · see citing works
Self-citationPark, C.-Y., Halevy, T., Lee, D. R., Sung, J. J., Lee, J. S., Yanuka, O., Benvenisty, N., & Kim, D.-W. (2015) · Cell Reports · doi:10.1016/j.celrep.2015.08.084 · Cited by 178 · see citing works
Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras
Samarasinghe, K. T. G., Jaime-Figueroa, S., Burgess, M., Nalawansha, D. A., Dai, K., Hu, Z., Bebenek, A., Holley, S. A., & Crews, C. M. (2021) · Cell Chemical Biology · doi:10.1016/j.chembiol.2021.03.011 · Cited by 182 · see citing works
Lau, C.-H., & Suh, Y. (2017) · F1000Research · doi:10.12688/f1000research.11243.1 · Cited by 161 · see citing works
Ortinski, P. I., O’Donovan, B., Dong, X., & Kantor, B. (2017) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2017.04.002 · Cited by 146 · see citing works
Genome-wide identification of CRISPR/Cas9 off-targets in human genome
Duan, J., Lu, G., Xie, Z., Lou, M., Luo, J., Guo, L., & Zhang, Y. (2014) · Cell Research · doi:10.1038/cr.2014.87 · Cited by 137 · see citing works
Engineering PGPMOs through Gene Editing and Systems Biology: A Solution for Phytoremediation?
Basu, S., Rabara, R. C., Negi, S., & Shukla, P. (2018) · Trends in Biotechnology · doi:10.1016/j.tibtech.2018.01.011 · Cited by 138 · see citing works
One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment
Grav, L. M., Lee, J. S., Gerling, S., Kallehauge, T. B., Hansen, A. H., Kol, S., Lee, G. M., Pedersen, L. E., & Kildegaard, H. F. (2015) · Biotechnology Journal · doi:10.1002/biot.201500027 · Cited by 131 · see citing works
Protospacer 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 · see citing works
Ojala, D. S., Sun, S., Santiago-Ortiz, J. L., Shapiro, M. G., Romero, P. A., & Schaffer, D. V. (2018) · Molecular Therapy · doi:10.1016/j.ymthe.2017.09.006 · Cited by 97 · see citing works
Tools for experimental and computational analyses of off-target editing by programmable nucleases
Bao, X. R., Pan, Y., Lee, C. M., Davis, T. H., & Bao, G. (2021) · Nature Protocols · doi:10.1038/s41596-020-00431-y · Cited by 106 · see citing works
Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: An overview
Martin, F., Sánchez-Hernández, S., Gutiérrez-Guerrero, A., Pinedo-Gomez, J., & Benabdellah, K. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17091507 · Cited by 92 · see citing works
“[11] used a bioinformatic approach to search for potential binding sites for gRNAs targeting the CCR5 gene, which is a target for the treatment of AIDS.”
MethodMolecular Therapies for Inherited Retinal Diseases—Current Standing, Opportunities and Challenges
Vázquez-Domínguez, I., Garanto, A., & Collin, R. W. J. (2019) · Genes · doi:10.3390/genes10090654 · Cited by 80 · see citing works
ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes
Miki, K., Deguchi, K., Nakanishi-Koakutsu, M., Lucena-Cacace, A., Kondo, S., Fujiwara, Y., Hatani, T., Sasaki, M., Naka, Y., Okubo, C., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-23816-3 · Cited by 76 · see citing works
Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches
Leal, A., Herreno-Pachón, A., Benincore-Flórez, E., Karunathilaka, A., & Tomatsu, S. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25052456 · Cited by 58 · see citing works
Tran, N. T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kühn, R., Rajewsky, K., & Chu, V. T. (2022) · Science Advances · doi:10.1126/sciadv.abm9106 · Cited by 49 · see citing works
Gao, Z., Harwig, A., Berkhout, B., & Herrera-Carrillo, E. (2017) · Transcription · doi:10.1080/21541264.2017.1322170 · Cited by 47 · see citing works
Concerns regarding ‘off-target’activity of genome editing endonucleases
Kadam, U. S., Shelake, R. M., Chavhan, R. L., & Suprasanna, P. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.03.027 · Cited by 47 · see citing works
Kulcsár, P. I., Tálas, A., Tóth, E., Nyeste, A., Ligeti, Z., Welker, Z., & Welker, E. (2020) · Nature Communications · doi:10.1038/s41467-020-15021-5 · Cited by 45 · see citing works
Massively targeted evaluation of therapeutic CRISPR off-targets in cells
Pan, X., Qu, K., Yuan, H., Xiang, X., Anthon, C., Pashkova, L., Liang, X., Han, P., Corsi, G. I., Xu, F., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-31543-6 · Cited by 42 · see citing works
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 · see citing works
crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays
Störtz, F., & Minary, P. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkaa885 · Cited by 37 · see citing works
Lohia, A., Sahel, D. K., Salman, M., Singh, V., Mariappan, I., Mittal, A., & Chitkara, D. (2022) · Asian Journal of Pharmaceutical Sciences · doi:10.1016/j.ajps.2022.02.001 · Cited by 33 · see citing works
Gene Therapy for Inherited Retinal Degeneration
Arbabi, A., Liu, A., & Ameri, H. (2019) · Journal of Ocular Pharmacology and Therapeutics · doi:10.1089/jop.2018.0087 · Cited by 30 · see citing works
Learning to quantify uncertainty in off-target activity for CRISPR guide RNAs
Özden, F., & Minary, P. (2024) · Nucleic Acids Research · doi:10.1093/nar/gkae759 · Cited by 21 · see citing works
Yaish, O., Asif, M., & Orenstein, Y. (2022) · Briefings in Bioinformatics · doi:10.1093/bib/bbac157 · Cited by 19 · see citing works
CRISPR off-target analysis platforms
Xu, C. L., Ruan, M. Z., Ragi, S. D., & Tsang, S. H. (2023) · Methods in molecular biology · doi:10.1007/978-1-0716-2651-1_26 · Cited by 6 · see citing works
Cas9-Induced Immune Response: A Potential Caution for Human Genome Editing
Zhang, C. C. (2019) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2019.10.025 · Cited by 4 · see citing works
- Authors
- Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., & Kim, J.-S.
- Affiliations
- Seoul National University (KR)
- Corresponding author
- Kim, Jin-Soo
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2013-11-19
- Volume
- 24
- Issue
- 1
- Pages
- 132-141
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC3875854
- OpenAlex
- W2105464583
- Cited by
- 1,356
- References
- 48
- Citations by year
- 2026: 14 · 2025: 50 · 2024: 76 · 2023: 77 · 2022: 94 · 2021: 125 · 2020: 125 · 2019: 122 · 2018: 126 · 2017: 150 · 2016: 160 · 2015: 115 · 2014: 117 · 2013: 4
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Scholarworks@UNIST (Ulsan National Institute of Science and Technology) (submittedVersion, cc-by-nc)
- Funders
- National Research Foundation
- National Research Foundation of Korea
- 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, RNA-guided endonuclease, off-target effects, Cas9 nickase, guide RNA design, single-strand breaks, targeted genome editing, chromosomal translocation, chromosomal deletion, Streptococcus pyogenes
- MeSH terms
- Base Sequence, Binding Sites, Chromosomes, Human, Deoxyribonuclease I, DNA, Endonucleases, HeLa Cells, Humans, Models, Genetic, Mutation, Genome, Human, K562 Cells, DNA Breaks, Double-Stranded, Genetic Loci, RNA, Small Untranslated, High-Throughput Nucleotide Sequencing, Exome, CRISPR-Cas Systems, CRISPR-Associated Proteins
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