The DOI matches its Crossref record; 29 of the 29 references it cites were found in a registry or an index.
DNA targeting specificity of RNA-guided Cas9 nucleases
Open accessAbstract
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
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
“For example, off-target cleavages of the Cas9 nucleases that cause oncogenic mutations in the genome have been reported extensively [123,124].”
From Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy (2022) · cites this paper as [123]
Experimental — accuracy not yet measuredThe 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 off-target sites are often sgRNA-dependent, since Cas9 is known to tolerate up to 3 mismatches between sgRNA and genomic DNA (Fu et al., 2013; Hsu et al., 2013; Wang et al., 2016a).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Although CRISPR/Cas systems exhibit tremendous potential in translational medicine, off-target effects remain a major challenge (Fu et al., 2013; Hsu et al., 2013; Pacesa et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“and it maintains cleaving activity even with 3-5 mismatches at the PAM-distal end of the gRNA molecule [59,81,102].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [102]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“The algorithm of MIT weights the position effect of the mismatches in the sgRNA to generate a score to evaluate off-target effects (Hsu et al., 2013; Haeussler et al., 2016).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
The cited source says what the claim attributes to it.
In the citing paper
“The PAM sequence adjacent to the target sequence greatly affects the editing efficiency and editing range of Cas9.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [2]
The cited source says what the claim attributes to it.
In the citing paper
“Cas9 is guided by a 20 nt fraction of the gRNA”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [102]
The check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“Recombinant expression and purification of EvoCas9–1 paired with chemically synthesized Evo-generated single guide RNA (sgRNA) exhibited comparable in vitro cleavage activity to SpCas9 paired with the canonical SpCas9 sgRNA”
From Sequence modeling and design from molecular to genome scale with Evo (2024) · cites this paper as [63]
Experimental — accuracy not yet measuredThe check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“When two sgRNAs are designed to simultaneously cut the two opposite strands of DNA within a small distance, DSBs can be created with markedly lower off-target effects (Hsu et al., 2013; Frock et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
Experimental — accuracy not yet measuredIn the citing paper
“By bioinformatic screening and efficiency/off-target prediction (14, 15), we identified four gRNA targets (protospacers; LTRs A-D) that avoid conserved transcription factor binding sites, minimizing the likelihood of altering host gene expression”
From RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection (2014) · cites this paper as [14]
In the citing paper
“as mentioned in several recent publications, Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells [12], [24], [25], [26], [27], thereby promoting undesired off-target mutations.”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [25]
In the citing paper
“Because the sequence specificity of Streptococcus pyogenes Cas9 tolerates 4 mismatches between the gRNA and complementary sequence”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [25]
In the citing paper
“non-specific cleavage may occur in the cellular genome [12], [24], [25], [26], [27].”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [25]
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 [91]
In the citing paper
“multiple mismatches between the guide RNA and its complementary target DNA sequence can be tolerated depending on the quantity, position, and base identity of mismatches [87,90,91,93], all of which contribute to potential off-target activity.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [91]
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 [91]
In the citing paper
“Collectively, these studies indicate that although each base within the 20 nt guide sequence contributes to overall specificity”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [91]
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
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
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
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
Self-citationEfficient 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
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
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
Self-citationA 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
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
Cited by
Sorted by citations
Items 301–400 of 1,041
CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells
Deng, W., Shi, X., Tjian, R., Lionnet, T., & Singer, R. H. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1515692112 · Cited by 279
Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell linePreprint
Essletzbichler, P., Konopka, T., Santoro, F., Chen, D., Gapp, B. V., Královics, R., Brummelkamp, T. R., Nijman, S. M. B., & Bürckstümmer, T. (2014) · Genome Research · doi:10.1101/gr.177220.114 · Cited by 277
Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner
Cano-Rodríguez, D., Gjaltema, R. A. F., Jilderda, L. J., Jellema, P. G., Dokter‐Fokkens, J., Ruiters, M. H. J., & Rots, M. G. (2016) · Nature Communications · doi:10.1038/ncomms12284 · Cited by 277
Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9
Shin, J. W., Kim, K., Chao, M. J., Atwal, R. S., Gillis, T., MacDonald, M. E., Gusella, J. F., & Lee, J. (2016) · Human Molecular Genetics · doi:10.1093/hmg/ddw286 · Cited by 294
Belmonte, J. C. I., Callaway, E. M., Caddick, S. J., Churchland, P. S., Feng, G., Homanics, G. E., Lee, K., Leopold, D. A., Miller, C. T., Mitchell, J. F., et al. (2015) · Neuron · doi:10.1016/j.neuron.2015.03.021 · Cited by 277
Self-citationOrthogonal gene knockout and activation with a catalytically active Cas9 nuclease
Dahlman, J. E., Abudayyeh, O. O., Joung, J., Gootenberg, J. S., Zhang, F., & Konermann, S. (2015) · Nature Biotechnology · doi:10.1038/nbt.3390 · Cited by 266
Self-citationA dynamic mode of mitotic bookmarking by transcription factors
Teves, S. S., An, L., Hansen, A. S., Xie, L., Darzacq, X., & Tjian, R. (2016) · eLife · doi:10.7554/elife.22280 · Cited by 274
Optical Control of CRISPR/Cas9 Gene Editing
Hemphill, J., Borchardt, E. K., Brown, K. A., Asokan, A., & Deiters, A. (2015) · Journal of the American Chemical Society · doi:10.1021/ja512664v · Cited by 276
CRISPR/cas systems redefine nucleic acid detection: Principles and methods
Wang, M., Zhang, R., & Li, J. (2020) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2020.112430 · Cited by 273
CRISPR/Cas9-Mediated Targeted Mutagenesis in the Liverwort Marchantia polymorpha L.
Sugano, S. S., Shirakawa, M., Takagi, J., Matsuda, Y., Shimada, T., Hara‐Nishimura, I., & Kohchi, T. (2014) · Plant and Cell Physiology · doi:10.1093/pcp/pcu014 · Cited by 269
Genome modification by CRISPR /Cas9
Ma, Y., Zhang, L., & Huang, X. (2014) · FEBS Journal · doi:10.1111/febs.13110 · Cited by 304
Jiang, W., Zhao, X., Gabrieli, T., Lou, C., Ebenstein, Y., & Zhu, T. F. (2015) · Nature Communications · doi:10.1038/ncomms9101 · Cited by 272
Braatz, J., Harloff, H., Mascher, M., Stein, N., Himmelbach, A., & Jung, C. (2017) · PLANT PHYSIOLOGY · doi:10.1104/pp.17.00426 · Cited by 283
Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus
Zhang, C., Meng, X., Wei, X., & Lü, L. (2015) · Fungal Genetics and Biology · doi:10.1016/j.fgb.2015.12.007 · Cited by 267
Stephan, T., Brüser, C., Deckers, M., Steyer, A. M., Balzarotti, F., Barbot, M., Behr, T. S., Heim, G., Hübner, W., Ilgen, P., et al. (2020) · The EMBO Journal · doi:10.15252/embj.2019104105 · Cited by 253
“Each gRNA was designed using the CRISPR design tool from Benchling based on the scoring models from (Hsu et al , 2013; Doench et al , 2016).”
Kang, X., He, W., Huang, Y., Qian, Y., Chen, Y., Gao, X., Sun, X., & Fan, Y. (2016) · Journal of Assisted Reproduction and Genetics · doi:10.1007/s10815-016-0710-8 · Cited by 271
Miyaoka, Y., Berman, J. R., Cooper, S., Mayerl, S. J., Chan, A. H., Zhang, B., Karlin-Neumann, G. A., & Conklin, B. R. (2016) · Scientific Reports · doi:10.1038/srep23549 · Cited by 277
Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A., & Ha, T. (2016) · Nature Communications · doi:10.1038/ncomms12778 · Cited by 269
Synthetic nucleases for genome engineering in plants: prospects for a bright future
Puchta, H., & Fauser, F. (2013) · The Plant Journal · doi:10.1111/tpj.12338 · Cited by 275
CRISPR technologies for genome, epigenome and transcriptome editing
Villiger, L., Joung, J., Koblan, L. W., Weissman, J. S., Abudayyeh, O. O., & Gootenberg, J. S. (2024) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-023-00697-6 · Cited by 280
“Cas9-dependent off-target editing can occur when the editor localizes to a similar off-target sequence binding in an sgRNA-dependent manner 110–113 .”
Zhu, X., Xu, Y., Yu, S., Lu, L., Ding, M., Cheng, J., Song, G., Gao, X., Yao, L., Fan, D., et al. (2014) · Scientific Reports · doi:10.1038/srep06420 · Cited by 264
Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein
Dong, D., Guo, M., Wang, S., Zhu, Y., Wang, S., Xiong, Z., Yang, J., Xu, Z., & Huang, Z. (2017) · Nature · doi:10.1038/nature22377 · Cited by 259
Therapeutic targeting of preleukemia cells in a mouse model of NPM1 mutant acute myeloid leukemia
Uckelmann, H. J., Kim, S. M., Wong, E. M. C., Hatton, C., Giovinazzo, H., Gadrey, J. Y., Krivtsov, A. V., Rücker, F. G., Döhner, K., McGeehan, G. M., et al. (2020) · Science · doi:10.1126/science.aax5863 · Cited by 267
Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans
Paix, A., Folkmann, A. W., & Seydoux, G. (2017) · Methods · doi:10.1016/j.ymeth.2017.03.023 · Cited by 247
Long, C., Li, H. Z., Tiburcy, M., Rodríguez-Caycedo, C., Kyrychenko, V., Zhou, H., Zhang, Y., Min, Y.-L., Shelton, J. M., Mammen, P. P. A., et al. (2018) · Science Advances · doi:10.1126/sciadv.aap9004 · Cited by 261
Multi‐input CRISPR/Cas genetic circuits that interface host regulatory networks
Nielsen, A. A. K., & Voigt, C. A. (2014) · Molecular Systems Biology · doi:10.15252/msb.20145735 · Cited by 259
Ždralević, M., Brand, A., Ianni, L. D., Dettmer, K., Reinders, J., Singer, K., Peter, K., Schnell, A., Bruß, C., Decking, S.-M., et al. (2018) · Journal of Biological Chemistry · doi:10.1074/jbc.ra118.004180 · Cited by 273
Precise genomic deletions using paired prime editing
Choi, J., Chen, W., Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza, R. M., Martin, B., & Shendure, J. (2021) · Nature Biotechnology · doi:10.1038/s41587-021-01025-z · Cited by 260
Ormond, K. E., Mortlock, D. P., Scholes, D. T., Bombard, Y., Brody, L. C., Faucett, W. A., Garrison, N. A., Hercher, L., Isasi, R., Middleton, A., et al. (2017) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2017.06.012 · Cited by 252
GuideScan software for improved single and paired CRISPR guide RNA design
Perez, A. R., Pritykin, Y., Vidigal, J. A., Chhangawala, S., Zamparo, L., Leslie, C. S., & Ventura, A. (2017) · Nature Biotechnology · doi:10.1038/nbt.3804 · Cited by 245
Wang, T., Guan, C., Guo, J., Liu, B., Wu, Y., Xie, Z., Zhang, C., & Xing, X. (2018) · Nature Communications · doi:10.1038/s41467-018-04899-x · Cited by 247
YB-1 regulates tiRNA-induced Stress Granule formation but not translational repression
Lyons, S. M., Achorn, C. A., Kedersha, N., Anderson, P., & Ivanov, P. V. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw418 · Cited by 255
The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli
Reisch, C. R., & Prather, K. L. J. (2015) · Scientific Reports · doi:10.1038/srep15096 · Cited by 250
Establishing RNA virus resistance in plants by harnessing CRISPR immune system
Zhang, T., Zheng, Q., Yi, X., An, H., Zhao, Y., Ma, S., & Zhou, G. (2018) · Plant Biotechnology Journal · doi:10.1111/pbi.12881 · Cited by 260
Identification of an ATP-sensitive potassium channel in mitochondria
Paggio, A., Checchetto, V., del Campo, A. F., Menabò, R., Marco, G. D., Lisa, F. D., Szabó, I., Rizzuto, R., & Stefani, D. D. (2019) · Nature · doi:10.1038/s41586-019-1498-3 · Cited by 255
Partial DNA-guided Cas9 enables genome editing with reduced off-target activity
Yin, H., Song, C.-Q., Suresh, S., Kwan, S.-Y., Wu, Q., Walsh, S., Ding, J., Bogorad, R. L., Zhu, L. J., Wolfe, S. A., et al. (2018) · Nature Chemical Biology · doi:10.1038/nchembio.2559 · Cited by 240
Genome Editing with Engineered Nucleases in Plants
Osakabe, Y., & Osakabe, K. (2014) · Plant and Cell Physiology · doi:10.1093/pcp/pcu170 · Cited by 253
Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer
Zhou, X., Xin, J., Fan, N., Zou, Q., Huang, J., Ouyang, Z., Zhao, Y., Zhao, B., Liu, Z., Lai, S., et al. (2014) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-014-1744-7 · Cited by 255
Yang, Z., Su, W., Wei, X., Qu, S., Zhao, D., Zhou, J., Wang, Y., Guan, Q., Qin, C., Xiang, J. Y., et al. (2023) · Cell Reports · doi:10.1016/j.celrep.2023.112945 · Cited by 261
Wu, Y., Zhou, H., Fan, X., Zhang, Y., Zhang, M., Wang, Y., Xie, Z., Bai, M., Yin, Q., Liang, D., et al. (2015) · Cell Research · doi:10.1038/cr.2014.160 · Cited by 247
Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies
Marino, N. D., Pinilla‐Redondo, R., Csörgő, B., & Bondy‐Denomy, J. (2020) · Nature Methods · doi:10.1038/s41592-020-0771-6 · Cited by 250
Cas9 versus Cas12a/Cpf1: Structure–function comparisons and implications for genome editing
Swarts, D. C., & Jinek, M. (2018) · WIREs RNA · doi:10.1002/wrna.1481 · Cited by 267
Potential pitfalls of CRISPR/Cas9‐mediated genome editing
Peng, R., Lin, G., & Li, J. (2016) · The FEBS Journal · doi:10.1111/febs.13586 · Cited by 213
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells
Lee, C. M., Cradick, T. J., & Bao, G. (2016) · Molecular Therapy · doi:10.1038/mt.2016.8 · Cited by 250
Self-citationIL6/STAT3 Signaling Hijacks Estrogen Receptor α Enhancers to Drive Breast Cancer Metastasis
Siersbæk, R., Scabia, V., Nagarajan, S., Chernukhin, I. V., Papachristou, E. K., Broome, R., Johnston, S. J., Joosten, S. E. P., Green, A. R., Kumar, S. S., et al. (2020) · Cancer Cell · doi:10.1016/j.ccell.2020.06.007 · Cited by 252
Torres-Ruiz, R., Martin, M. C., Garcia, A. F. R., Cigudosa, J. C., Ramı́rez, J. C., & Rodríguez, S. (2014) · Nature Communications · doi:10.1038/ncomms4964 · Cited by 255
Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression
Luo, M. L., Mullis, A. S., Leenay, R. T., & Beisel, C. L. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku971 · Cited by 247
Review of CRISPR/Cas9 sgRNA Design Tools
Cui, Y., Xu, J., Cheng, M., Liao, X., & Peng, S. (2018) · Interdisciplinary Sciences Computational Life Sciences · doi:10.1007/s12539-018-0298-z · Cited by 253
Wang, Y., Wang, S., Chen, W., Song, L., Zhang, Y., Shen, Z., Yu, F., Li, M., & Ji, Q. (2018) · Applied and Environmental Microbiology · doi:10.1128/aem.01834-18 · Cited by 251
Konstantakos, V., Nentidis, A., Krithara, A., & Παλιούρας, Γ. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac192 · Cited by 263
“(54) reported that high concentrations of the SpCas9–sgRNA complex increase offsite target effects, whereas lower concentrations of Cas9 increase specificity and reduce on-target cleavage activity.”
BackgroundCRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells
Zhang, Y., Zhang, X., Cheng, C., Mu, W., Liu, X., Li, N., Wei, X., Liu, X., Xia, C., & Wang, H. (2017) · Frontiers of Medicine · doi:10.1007/s11684-017-0543-6 · Cited by 239
Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects
Gao, Y., Wu, H., Wang, Y., Liu, X., Chen, L., Li, Q., Cui, C., Liu, X., Zhang, J., & Zhang, Y. (2017) · Genome biology · doi:10.1186/s13059-016-1144-4 · Cited by 246
Li, Y., Cao, X., Zhu, Y. L., Yang, X., Zhang, K., Xiao, Z., Wang, H., Zhao, J., Zhang, L., Li, G., et al. (2019) · New Phytologist · doi:10.1111/nph.15678 · Cited by 231
Self-citationSelection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing
Liang, G., Zhang, H., Lou, D., & Yu, D. (2016) · Scientific Reports · doi:10.1038/srep21451 · Cited by 240
The New State of the Art: Cas9 for Gene Activation and Repression
Russa, M. F. L., & Qi, L. S. (2015) · Molecular and Cellular Biology · doi:10.1128/mcb.00512-15 · Cited by 241
Xu, R., Li, H., Qin, R., Li, J., Qiu, C.-H., Yang, Y.-C., Ma, H., Li, L., Wei, P., & Yang, J.-B. (2015) · Scientific Reports · doi:10.1038/srep11491 · Cited by 227
Minimal PAM specificity of a highly similar SpCas9 ortholog
Chatterjee, P., Jakimo, N., & Jacobson, J. M. (2018) · Science Advances · doi:10.1126/sciadv.aau0766 · Cited by 229
A CRISPR view of developmentPreprint
Harrison, M. M., Jenkins, B., O’Connor-Giles, K. M., & Wildonger, J. (2014) · Genes & Development · doi:10.1101/gad.248252.114 · Cited by 234
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
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
Targeted mutagenesis using CRISPR/Cas system in medaka
Ansai, S., & Kinoshita, M. (2014) · Biology Open · doi:10.1242/bio.20148177 · Cited by 227
Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice
Xu, R., Li, H., Qin, R., Wang, L., Li, L., Wei, P., & Yang, J. (2014) · Rice · doi:10.1186/s12284-014-0005-6 · Cited by 232
Efficient CRISPR-Cas9–mediated genome editing in Plasmodium falciparum
Wagner, J. C., Platt, R. J., Goldfless, S. J., Zhang, F., & Niles, J. C. (2014) · Nature Methods · doi:10.1038/nmeth.3063 · Cited by 227
Self-citationKon, S., Ishibashi, K., Katoh, H., Kitamoto, S., Shirai, T., Tanaka, S., Kajita, M., Ishikawa, S., Yamauchi, H., Yako, Y., et al. (2017) · Nature Cell Biology · doi:10.1038/ncb3509 · Cited by 226
CRISPR‐Cas9 Genome Editing in Drosophila
Gratz, S. J., Rubinstein, C. D., Harrison, M. M., Wildonger, J., & O’Connor-Giles, K. M. (2015) · Current Protocols in Molecular Biology · doi:10.1002/0471142727.mb3102s111 · Cited by 214
Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes
Temoche-Diaz, M. M., Shurtleff, M. J., Nottingham, R. M., Yao, J., Fadadu, R. P., Lambowitz, A. M., & Schekman, R. (2019) · eLife · doi:10.7554/elife.47544 · Cited by 227
Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease
Fujii, W., Kawasaki, K., Sugiura, K., & Naito, K. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt772 · Cited by 227
Application of CRISPR/Cas9 in plant biology
Liu, X., Wu, S., Xu, J., Sui, C., & Wei, J. (2017) · Acta Pharmaceutica Sinica B · doi:10.1016/j.apsb.2017.01.002 · Cited by 235
Chen, W., McKenna, A., Schreiber, J., Haeussler, M., Yin, Y., Agarwal, V., Noble, W. S., & Shendure, J. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz487 · Cited by 216
Michels, B. E., Mosa, M. H., Streibl, B. I., Zhan, T., Menche, C., Abou-El-Ardat, K., Darvishi, T., Członka, E., Wagner, S., Winter, J., et al. (2020) · Cell Stem Cell · doi:10.1016/j.stem.2020.04.003 · Cited by 222
Schiroli, G., Ferrari, S., Conway, A., Jacob, A., Capo, V., Albano, L., Plati, T., Castiello, M. C., Sanvito, F., Gennery, A. R., et al. (2017) · Science Translational Medicine · doi:10.1126/scitranslmed.aan0820 · Cited by 214
Li, C., Guan, X., Du, T., Jin, W., Wu, B., Liu, Y., Wang, P., Hu, B., Griffin, G. E., Shattock, R. J., et al. (2015) · Journal of General Virology · doi:10.1099/vir.0.000139 · Cited by 192
CRISPR-Cas9 nuclear dynamics and target recognition in living cells
Ma, H., Tu, L., Naseri, A., Huisman, M., Zhang, S., Grünwald, D., & Pederson, T. (2016) · The Journal of Cell Biology · doi:10.1083/jcb.201604115 · Cited by 224
Animal models of arrhythmia: classic electrophysiology to genetically modified large animals
Clauß, S., Bleyer, C., Schüttler, D., Tomsits, P., Renner, S., Klymiuk, N., Wakili, R., Maßberg, S., Wolf, E., & Kääb, S. (2019) · Nature Reviews Cardiology · doi:10.1038/s41569-019-0179-0 · Cited by 233
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
CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation
Tuladhar, R., Yeu, Y., Tyler Piazza, J., Tan, Z., Rene Clemenceau, J., Wu, X., Barrett, Q., Herbert, J., Mathews, D. H., Kim, J., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-12028-5 · Cited by 219
Self-citationRapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system
Inui, M., Miyado, M., Igarashi, M., Tamano, M., Kubo, A., Yamashita, S., Asahara, H., Fukami, M., & Takada, S. (2014) · Scientific Reports · doi:10.1038/srep05396 · Cited by 230
CRISPR/Cas9 advances engineering of microbial cell factories
Jakočiūnas, T., Jensen, M. K., & Keasling, J. D. (2015) · Metabolic Engineering · doi:10.1016/j.ymben.2015.12.003 · Cited by 225
Esposito, R., Bosch, N., Lanzós, A., Polidori, T., Pulido-Quetglas, C., & Johnson, R. (2019) · Cancer Cell · doi:10.1016/j.ccell.2019.01.019 · Cited by 223
Optimization of AsCas12a for combinatorial genetic screens in human cells
DeWeirdt, P. C., Sanson, K. R., Sangree, A. K., Hegde, M., Hanna, R. E., Feeley, M. N., Griffith, A. L., Teng, T., Borys, S. M., Strand, C., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0600-6 · Cited by 215
Cao, J. P., Wu, L., Zhang, S., Lu, M., Cheung, W. K. C., Cai, W. L., Gale, M., Xu, Q., & Yan, Q. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw660 · Cited by 218
Isolated polycystic liver disease genes define effectors of polycystin-1 function
Besse, W., Dong, K., Choi, J., Punia, S., Fedeles, S. V., Choi, M., Gallagher, A.-R., Huang, E. B., Gulati, A., Knight, J., et al. (2017) · Journal of Clinical Investigation · doi:10.1172/jci90129 · Cited by 203
Liang, C., Li, F., Wang, L., Zhang, Z., Wang, C., He, B., Li, J., Chen, Z., Shaikh, A. B., Liu, J., et al. (2017) · Biomaterials · doi:10.1016/j.biomaterials.2017.09.015 · Cited by 217
Self-citationCRISPR off-target analysis in genetically engineered rats and mice
Anderson, K. R., Haeussler, M., Watanabe, C. K., Janakiraman, V., Lund, J. M., Modrusan, Z. D., Stinson, J., Bei, Q., Buechler, A., Yu, C., et al. (2018) · Nature Methods · doi:10.1038/s41592-018-0011-5 · Cited by 210
Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
Endo, M., Mikami, M., & Toki, S. (2014) · Plant and Cell Physiology · doi:10.1093/pcp/pcu154 · Cited by 216
Application of CRISPR-Cas9 genome editing technology in various fields: A review
Ansori, A. N. M., Antonius, Y., Susilo, R. J. K., Hayaza, S., Kharisma, V. D., Parikesit, A. A., Zainul, R., Jakhmola, V., Saklani, T., Ребезов, М. Б., et al. (2023) · Narra J · doi:10.52225/narra.v3i2.184 · Cited by 243
“By using CRISPR-Cas9 to target and control gene regulatory elements, such as promoters or enhancers, researchers can gain insights into the roles of specific genes and their interactions [7].”
MethodCRISPR/Cas9 and Genome Editing in Drosophila
Bassett, A. R., & Liu, J. (2013) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2013.12.004 · Cited by 216
Zuo, E., Cai, Y., Li, K., Yu, W., Wang, B.-A., Sun, Y., Liu, Z., Liu, J., Hu, X., Wei, W., et al. (2017) · Cell Research · doi:10.1038/cr.2017.81 · Cited by 201
Design and analysis of CRISPR–Cas experiments
Hanna, R. E., & Doench, J. G. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0490-7 · Cited by 216
Distinct subnetworks of the thalamic reticular nucleus
Li, Y., López-Huerta, V. G., Adiconis, X., Levandowski, K. M., Choi, S., Simmons, S., Arias-García, M. A., Guo, B., Yao, A. Y., Blosser, T. R., et al. (2020) · Nature · doi:10.1038/s41586-020-2504-5 · Cited by 211
Self-citationMyosin-II mediated traction forces evoke localized Piezo1-dependent Ca2+ flickers
Ellefsen, K. L., Holt, J. R., Chang, A. C., Nourse, J. L., Arulmoli, J., Mekhdjian, A. H., Abuwarda, H., Tombola, F., Flanagan, L. A., Dunn, A. R., et al. (2019) · Communications Biology · doi:10.1038/s42003-019-0514-3 · Cited by 217
Structural basis for Cas9 off-target activity
Pacesa, M., Lin, C.-H., Cléry, A., Saha, A., Arantes, P. R., Bargsten, K., Irby, M. J., Allain, F. H.-T., Palermo, G., Cameron, P., et al. (2022) · Cell · doi:10.1016/j.cell.2022.09.026 · Cited by 198
Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes
Oliveros, J. C., Franch, M., Tabas‐Madrid, D., León, D. S., Montoliu, L., Cubas, P., & Pazos, F. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw407 · Cited by 204
Lamano, J. B., Lamano, J. B., Li, Y. D., DiDomenico, J. D., Choy, W., Veliceasa, D., Oyon, D. E., Fakurnejad, S., Ampie, L., Kesavabhotla, K., et al. (2019) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-18-2402 · Cited by 213
Self-citationGenome-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
Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens
Morris, J. A., Caragine, C., Daniloski, Z., Domingo, J., Barry, T., Lu, L., Davis, K., Ziosi, M., Glinos, D. A., Hao, S., et al. (2023) · Science · doi:10.1126/science.adh7699 · Cited by 208
“These gRNAs were optimizedforminimaloff-target activity( 21 , 22 ).”
CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems
Jiang, W., & Marraffini, L. A. (2015) · Annual Review of Microbiology · doi:10.1146/annurev-micro-091014-104441 · Cited by 219
Self-citationSauer, N. J., Narváez-Vásquez, J., Mozoruk, J., Miller, R. B., Warburg, Z. J., Woodward, M. J., Mihiret, Y. A., Lincoln, T. A., Segami, R. E., Sanders, S. L., et al. (2016) · PLANT PHYSIOLOGY · doi:10.1104/pp.15.01696 · Cited by 207
Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9
Koo, T., Lee, J., & Kim, J. (2015) · Molecules and Cells · doi:10.14348/molcells.2015.0103 · Cited by 213
Simple and Rapid In Vivo Generation of Chromosomal Rearrangements using CRISPR/Cas9 Technology
Blasco, R. B., Karaca, E., Ambrogio, C., Cheong, T.-C., Karayol, E., Minero, V. G., Voena, C., & Chiarle, R. (2014) · Cell Reports · doi:10.1016/j.celrep.2014.10.051 · Cited by 210
Metadata
- Authors
- 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., Cradick, T. J., Marraffini, L. A., Bao, G., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Georgia Institute of Technology (US)
- Koch Institute for Integrative Cancer Research At MIT (US)
- Rockefeller University (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2013-07-21
- Volume
- 31
- Issue
- 9
- Pages
- 827-832
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3969858
- OpenAlex
- W2036176224
- Cited by
- 4,741
- References
- 29
- Citations by year
- 2026: 242 · 2025: 300 · 2024: 304 · 2023: 331 · 2022: 372 · 2021: 424 · 2020: 416 · 2019: 422 · 2018: 463 · 2017: 438 · 2016: 402 · 2015: 297 · 2014: 269 · 2013: 53
- FWCI
- 152.56
- 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)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion)
- PMC
- Funders
- National Science Foundation
- Howard Hughes Medical Institute
- Damon Runyon Cancer Research Foundation
- McKnight Foundation
- Life Sciences Research Foundation
- National Institutes of Health (R01‐grant, GM34277)
- Harvard Stem Cell Institute
- National Institute of Mental Health (DP1MH100706)
- National Eye Institute (PN2EY018244)
- National Cancer Institute (R01CA133404)
- National Institute of General Medical Sciences (R01GM034277, R01 GM34277, T32GM007753)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- National Center for Chronic Disease Prevention and Health Promotion (DP1‑MH100706)
- 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
- SpCas9, single guide RNA, genome editing, off-target effects, mismatch tolerance, DNA methylation, indel mutations, guide RNA design, target site selection, HEK293T cells, web-based design tool
- MeSH terms
- Bacterial Proteins, Base Sequence, Deoxyribonucleases, DNA, Genetic Engineering, Molecular Sequence Data, Streptococcus pyogenes, Transcription Factors, Base Pair Mismatch, RNA, Small Untranslated
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
