Mapping the genomic landscape of CRISPR-Cas9 cleavage
Abstract
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
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?
SITE-seq reduces the background noise of Digenome-seq and requires much less sequencing coverage (~0.62-2.46 million reads for human genome).
“SITE-seq reduces the background noise of Digenome-seq and requires much less sequencing coverage (∼0.62–2.46 million reads for human genome).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cameron et al., 2017
Cites
The Sequence Alignment/Map format and SAMtools
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., & 1000 Genome Project Data Processing Subgroup (2009) · Bioinformatics · doi:10.1093/bioinformatics/btp352 · Cited by 62,041 · see citing works
Fast gapped-read alignment with Bowtie 2
Langmead, B., & Salzberg, S. L. (2012) · Nature Methods · doi:10.1038/nmeth.1923 · Cited by 56,400 · see citing works
Model-based Analysis of ChIP-Seq (MACS)
Zhang, Y., Liu, T., Meyer, C. A., Eeckhoute, J., Johnson, D. S., Bernstein, B. E., Nusbaum, C., Myers, R. M., Brown, M., Li, W., et al. (2008) · Genome Biology · doi:10.1186/gb-2008-9-9-r137 · Cited by 18,386 · see citing works
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
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
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
Development and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762 · see citing works
Fitting a mixture model by expectation maximization to discover motifs in biopolymers.
Bailey, T. L., & Elkan, C. (1994) · PubMed · Cited by 4,933 · 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
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
Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1208507109 · Cited by 2,769 · see citing works
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339 · see citing works
Crystal structure of Cas9 in complex with guide RNA and target DNA
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014) · Cell · doi:10.1016/j.cell.2014.02.001 · Cited by 2,306 · see citing works
GUIDE-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
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
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 3× · see citing works
“Of note, the on-target site was recovered with the lowest concentration of nuclease that permitted recovery of target sites, most often at 0.25 nM sgRNP ( Supplementary Tables 2 – 9 ).”
BackgroundInfluentialStructural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease
Anders, C., Niewoehner, O., Duerst, A., & Jínek, M. (2014) · Nature · doi:10.1038/nature13579 · Cited by 1,319 · see citing works
CCTop: An intuitive, flexible and reliable CRISPR/Cas9 target prediction tool
Stemmer, M., Thumberger, T., del Sol Keyer, M., Wittbrodt, J., & Mateo, J. L. (2015) · PLoS ONE · doi:10.1371/journal.pone.0124633 · Cited by 1,088 · 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
Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection
Liang, X., Potter, J., Kumar, S., Zou, Y., Quintanilla, R., Sridharan, M., Carte, J., Chen, W., Roark, N., Ranganathan, S., et al. (2015) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2015.04.024 · Cited by 733 · see citing works
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
Frock, R. L., Hu, J., Meyers, R. M., Ho, Y.-J., Kii, E., & Alt, F. W. (2015) · Nature Biotechnology · doi:10.1038/nbt.3101 · Cited by 673 · see citing works
Check your cultures! A list of cross‐contaminated or misidentified cell lines
Capes‐Davis, A., Theodosopoulos, G., Atkin, I., Drexler, H. G., Kohara, A., MacLeod, R. A. F., Masters, J. R., Nakamura, Y., Reid, Y. A., Reddel, R. R., et al. (2010) · International Journal of Cancer · doi:10.1002/ijc.25242 · Cited by 520 · cited 1× · see citing works
“HEK cells are listed in the database of commonly misidentified cell lines 34 , and they were chosen in this study because previous experiments showed that they support robust gene editing 28 .”
MethodNucleotide-resolution DNA double-strand break mapping by next-generation sequencing
Crosetto, N., Mitra, A., Silva, M. J., Bienko, M., Dojer, N., Wang, Q., Karaca, E., Chiarle, R., Skrzypczak, M., Ginalski, K., et al. (2013) · Nature Methods · doi:10.1038/nmeth.2408 · Cited by 465 · 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
Nucleosomes impede Cas9 access to DNA in vivo and in vitro
Horlbeck, M. A., Witkowsky, L. B., Guglielmi, B., Replogle, J. M., Gilbert, L. A., Villalta, J. E., Torigoe, S. E., Tjian, R., & Weissman, J. S. (2016) · eLife · doi:10.7554/elife.12677 · Cited by 415 · see citing works
Expanding the Biologist’s Toolkit with CRISPR-Cas9
Sternberg, S. H., & Doudna, J. A. (2015) · Molecular Cell · doi:10.1016/j.molcel.2015.02.032 · Cited by 398 · see citing works
F-Seq: a feature density estimator for high-throughput sequence tags
Boyle, A. P., Guinney, J., Crawford, G. E., & Furey, T. S. (2008) · Bioinformatics · doi:10.1093/bioinformatics/btn480 · Cited by 363 · see citing works
Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality
Briner, A. E., Donohoue, P. D., Gomaa, A. A., Selle, K., Slorach, E. M., Nye, C. H., Haurwitz, R. E., Beisel, C. L., May, A. P., & Barrangou, R. (2014) · Molecular Cell · doi:10.1016/j.molcel.2014.09.019 · Cited by 251 · see citing works
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 · see citing works
Blitzblau, H. G., Bell, G. W., Rodriguez, J., Bell, S. P., & Hochwagen, A. (2007) · Current Biology · doi:10.1016/j.cub.2007.10.066 · Cited by 167 · see citing works
Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes
Richardson, C. D., Ray, G. J., Bray, N., & Corn, J. E. (2016) · Nature Communications · doi:10.1038/ncomms12463 · Cited by 80 · see citing works
Distinct patterns of Cas9 mismatch tolerancein vitroandin vivo
Fu, B. X. H., St.Onge, R. P., Fire, A. Z., & Smith, J. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw417 · Cited by 71 · see citing works
Landscape of target:guide homology effects on Cas9-mediated cleavage
Fu, B. X. H., Hansen, L. L., Artiles, K. L., Nonet, M. L., & Fire, A. Z. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku1102 · Cited by 66 · see citing works
SITE-Seq: A Genome-wide Method to Measure Cas9 Cleavage
Cameron, P., Cameron, P., Settle, A. H., Fuller, C. K., Thompson, M. S., Cigan, A. M., Young, J. K., & May, A. P. (2017) · Protocol Exchange · doi:10.1038/protex.2017.043 · Cited by 16 · see citing works
Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis
Gillmore, J. D., Gane, E., Taubel, J., Kao, J., Fontana, M., Maitland, M. L., Seitzer, J., O’Connell, D., Walsh, K. R., Wood, K., et al. (2021) · New England Journal of Medicine · doi:10.1056/nejmoa2107454 · Cited by 1,766 · see citing works
Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., et al. (2018) · Nature Medicine · doi:10.1038/s41591-018-0137-0 · Cited by 782 · see citing works
Off-target effects in CRISPR/Cas9 gene editing
Guo, C., Ma, X., Gao, F., & Guo, Y. (2023) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1143157 · Cited by 625 · see citing works
“To reduce such cost, scientists developed SITE-seq, a method that adds a selective biotinylation reaction on the cleaved genomic sites and leverages streptavidin pulldown to enrich these sites before sequencing (Cameron et al., 2017).”
MethodPast, present, and future of CRISPR genome editing technologies
Pacesa, M., Pelea, O., & Jinek, M. (2024) · Cell · doi:10.1016/j.cell.2024.01.042 · Cited by 609 · see citing works
CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors
Wang, D., Zhang, F., & Gao, G. (2020) · Cell · doi:10.1016/j.cell.2020.03.023 · Cited by 526 · 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
Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Wienert, B., Wyman, S. K., Richardson, C. D., Yeh, C. D., Akcakaya, P., Porritt, M. J., Morlock, M., Vu, J. T., Kazane, K. R., Watry, H. L., et al. (2019) · Science · doi:10.1126/science.aav9023 · Cited by 434 · see citing works
Manghwar, H., Li, B., Ding, X. L., Hussain, A., Lindsey, K., Zhang, X., & Jin, S. (2020) · Advanced Science · doi:10.1002/advs.201902312 · Cited by 394 · see citing works
Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs
Listgarten, J., Weinstein, M., Kleinstiver, B. P., Sousa, A. A., Joung, J. K., Crawford, J., Gao, K., Hoang, L., Elibol, M., Doench, J. G., et al. (2018) · Nature Biomedical Engineering · doi:10.1038/s41551-017-0178-6 · Cited by 336 · see citing works
Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis.
Wang, X., Wu, X., Tan, B.-H., Zhu, L., Zhang, Y., Lin, L., Xiao, Y., Sun, A., Wan, X., Liu, S.-Y., et al. (2024) · Cell · doi:10.1016/j.cell.2024.06.027 · Cited by 302 · see citing works
Tang, X. D., Liu, G., Zhou, J., Ren, Q., You, Q., Tian, L., Xin, X., Zhong, Z., Liu, B., Zheng, X., et al. (2018) · Genome biology · doi:10.1186/s13059-018-1458-5 · Cited by 305 · see citing works
CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity
Lazzarotto, C. R., Malinin, N. L., Li, Y., Zhang, R., Yang, Y., Lee, G., Cowley, E., He, Y., Lan, X., Jividen, K., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0555-7 · Cited by 283 · see citing works
An, M., Raguram, A., Du, S. W., Banskota, S., Davis, J. R., Newby, G. A., Chen, P., Palczewski, K., & Liu, D. R. (2024) · Nature Biotechnology · doi:10.1038/s41587-023-02078-y · Cited by 275 · see citing works
Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering
Wada, N., Ueta, R., Osakabe, Y., & Osakabe, K. (2020) · BMC Plant Biology · doi:10.1186/s12870-020-02385-5 · Cited by 271 · see citing works
Vaidyanathan, S., Azizian, K. T., Haque, A. K. M. A., Henderson, J. M., Hendel, A., Shore, S., Antony, J. S., Hogrefe, R. I., Kormann, M. S. D., Porteus, M. H., et al. (2018) · Molecular Therapy Nucleic Acids · doi:10.1016/j.omtn.2018.06.010 · Cited by 262 · see citing works
Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq
Turchiano, G., Andrieux, G., Klermund, J., Blattner, G., Pennucci, V., el Gaz, M., Monaco, G., Poddar, S., Mussolino, C., Cornu, T. I., et al. (2021) · Cell stem cell · doi:10.1016/j.stem.2021.02.002 · Cited by 231 · see citing works
CRISPR 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 207 · see citing works
Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Tao, J., Bauer, D. E., & Chiarle, R. (2023) · Nature Communications · doi:10.1038/s41467-023-35886-6 · Cited by 215 · see citing works
Li, J., Manghwar, H., Sun, L., Wang, P., Wang, G., Sheng, H., Zhang, J., Liu, H., Qin, L., Rui, H., et al. (2018) · Plant Biotechnology Journal · doi:10.1111/pbi.13020 · Cited by 205 · see citing works
Computational approaches for effective CRISPR guide RNA design and evaluation
Liu, G., Zhang, Y., & Zhang, T. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.11.006 · Cited by 199 · see citing works
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 · see citing works
Self-citationHighly Parallel Profiling of Cas9 Variant Specificity
Schmid-Burgk, J. L., Gao, L., Li, D., Gardner, Z., Strecker, J., Lash, B., & Zhang, F. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.02.023 · Cited by 197 · see citing works
Png, Y. T., Vinanica, N., Kamiya, T., Shimasaki, N., Coustan‐Smith, E., & Campana, D. (2017) · Blood Advances · doi:10.1182/bloodadvances.2017009928 · Cited by 189 · see citing works
Höijer, I., Emmanouilidou, A., Östlund, R., van Schendel, R., Bozorgpana, S., Tijsterman, M., Feuk, L., Gyllensten, U. B., den Hoed, M., & Ameur, A. (2022) · Nature Communications · doi:10.1038/s41467-022-28244-5 · Cited by 189 · see citing works
The CRISPR/Cas9 system and its applications in crop genome editing
Bao, A., Burritt, D. J., Chen, H., Zhou, X., Cao, D., & Tran, L.-S. P. (2019) · Critical Reviews in Biotechnology · doi:10.1080/07388551.2018.1554621 · Cited by 185 · see citing works
Development of CRISPR-Cas systems for genome editing and beyond
Zhang, F. (2019) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583519000052 · Cited by 185 · see citing works
CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease
Sharma, A., Boelens, J. J., Cancio, M., Hankins, J. S., Bhad, P. A., Azizy, M., Lewandowski, A., Zhao, X., Chitnis, S. D., Peddinti, R., et al. (2023) · New England Journal of Medicine · doi:10.1056/nejmoa2215643 · Cited by 186 · see citing works
R-loop formation and conformational activation mechanisms of Cas9
Pačesa, M., Loeff, L., Querques, I., Muckenfuss, L. M., Sawicka, M., & Jínek, M. (2022) · Nature · doi:10.1038/s41586-022-05114-0 · Cited by 166 · 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
CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters
Alkan, F., Wenzel, A., Anthon, C., Havgaard, J. H., & Gorodkin, J. (2018) · Genome biology · doi:10.1186/s13059-018-1534-x · Cited by 167 · see citing works
Lau, C.-H., & Suh, Y. (2017) · F1000Research · doi:10.12688/f1000research.11243.1 · Cited by 161 · see citing works
Unbiased investigation of specificities of prime editing systems in human cells
Kim, D. Y., Moon, S. B., Ko, J., Kim, Y.-S., & Kim, D. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa764 · Cited by 154 · see citing works
Genome-wide profiling of adenine base editor specificity by EndoV-seq
Liang, P., Xie, X., Zhi, S., Sun, H., Zhang, X., Chen, Y., Chen, Y., Xiong, Y., Ma, W., Liu, D., et al. (2019) · Nature Communications · doi:10.1038/s41467-018-07988-z · Cited by 148 · see citing works
DNA stretching induces Cas9 off-target activity
Newton, M. D., Taylor, B., Driessen, R. P. C., Roos, L., Cvetešić, N., Allyjaun, S., Lenhard, B., Cuomo, M. E., & Rueda, D. (2019) · Nature Structural & Molecular Biology · doi:10.1038/s41594-019-0188-z · Cited by 134 · see citing works
Zhang, D., Zhang, H., Li, T., Chen, K., Qiu, J., & Gao, C. (2017) · Genome biology · doi:10.1186/s13059-017-1325-9 · Cited by 133 · see citing works
Genetic Biocontrol for Invasive Species
Teem, J. L., Alphey, L., Descamps, S., Edgington, M. P., Edwards, O. R., Gemmell, N. J., Harvey‐Samuel, T., Melnick, R. L., Oh, K. P., Piaggio, A. J., et al. (2020) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2020.00452 · Cited by 134 · see citing works
Computational Tools and Resources for CRISPR/Cas Genome Editing
Li, C., Chu, W., Gill, R. A., Sang, S., Shi, Y., Hu, X., Yang, Y., Zaman, Q. U., & Zhang, B. (2022) · Genomics Proteomics & Bioinformatics · doi:10.1016/j.gpb.2022.02.006 · Cited by 131 · see citing works
The NIH Somatic Cell Genome Editing program
Saha, K., Sontheimer, E. J., Brooks, P. J., Dwinell, M. R., Gersbach, C. A., Liu, D. R., Murray, S. A., Tsai, S. Q., Wilson, R. C., Anderson, D. G., et al. (2021) · Nature · doi:10.1038/s41586-021-03191-1 · Cited by 125 · see citing works
The Current State and Future of CRISPR-Cas9 gRNA Design Tools
Wilson, L. O. W., O’Brien, A. R., & Bauer, D. C. (2018) · Frontiers in Pharmacology · doi:10.3389/fphar.2018.00749 · Cited by 124 · see citing works
NmeCas9 is an intrinsically high-fidelity genome-editing platform
Amrani, N., Gao, X. D., Liu, P., Edraki, A., Mir, A., Ibraheim, R., Gupta, A., Sasaki, K. E., Wu, T., Donohoue, P. D., et al. (2018) · Genome biology · doi:10.1186/s13059-018-1591-1 · Cited by 124 · see citing works
Self-citationDesign of highly functional genome editors by modelling CRISPR–Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J. P., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pačesa, M., et al. (2025) · Nature · doi:10.1038/s41586-025-09298-z · Cited by 212 · see citing works
Self-citationEvaluation and Reduction of CRISPR Off-Target Cleavage Events
Vakulskas, C. A., & Behlke, M. A. (2019) · Nucleic Acid Therapeutics · doi:10.1089/nat.2019.0790 · Cited by 121 · see citing works
A new age of precision gene therapy
Schambach, A., Buchholz, C. J., Torres-Ruiz, R., Cichutek, K., Morgan, M. A., Trapani, I., & Büning, H. (2023) · The Lancet · doi:10.1016/s0140-6736(23)01952-9 · Cited by 125 · see citing works
Singh, M., Kumar, M., Albertsen, M. C., Young, J. K., & Cigan, A. M. (2018) · Plant Molecular Biology · doi:10.1007/s11103-018-0749-2 · Cited by 116 · see citing works
Self-citationHybridization Kinetics Explains CRISPR-Cas Off-Targeting Rules
Klein, M., Eslami-Mossallam, B., Arroyo, D. G., & Depken, M. (2018) · Cell Reports · doi:10.1016/j.celrep.2018.01.045 · Cited by 107 · see citing works
Harnessing type I CRISPR–Cas systems for genome engineering in human cells
Cameron, P., Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, P. D., Rotstein, T., Kohrs, B., Nyer, D. B., et al. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0310-0 · Cited by 114 · see citing works
Self-citationLessard, S., Francioli, L. C., Alföldi, J., Tardif, J., Ellinor, P. T., MacArthur, D. G., Lettre, G., Orkin, S. H., & Canver, M. C. (2017) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1714640114 · Cited by 112 · see citing works
Sherkatghanad, Z., Abdar, M., Charlier, J., & Makarenkov, V. (2023) · Briefings in Bioinformatics · doi:10.1093/bib/bbad131 · Cited by 117 · see citing works
DIG-Seq: A genome-wide CRISPR off-target profiling method using chromatin DNA
Kim, D., & Kim, J.-S. (2018) · Genome Research · doi:10.1101/gr.236620.118 · Cited by 108 · see citing works
Optimizing genome editing strategy by primer-extension-mediated sequencing
Yin, J., Liu, M., Liu, Y., Wu, J., Gan, T., Zhang, W., Li, Y., Zhou, Y., & Hu, J. (2019) · Cell Discovery · doi:10.1038/s41421-019-0088-8 · Cited by 103 · 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
Novel features and considerations for ERA and regulation of crops produced by genome editing
Duensing, N., Sprink, T., Parrott, W. A., Fedorova, M., Lema, M. A., Wolt, J. D., & Bartsch, D. (2018) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2018.00079 · Cited by 106 · see citing works
Template plasmid integration in germline genome-edited cattle
Norris, A. L., Lee, S. S., Greenlees, K. J., Tadesse, D. A., Miller, M. F., & Lombardi, H. A. (2020) · Nature Biotechnology · doi:10.1038/s41587-019-0394-6 · Cited by 104 · see citing works
Lin, J., Zhang, Z., Zhang, S., Chen, J., & Wong, K. (2020) · Advanced Science · doi:10.1002/advs.201903562 · Cited by 101 · see citing works
Eckerstorfer, M. F., Dolezel, M., Heissenberger, A., Miklau, M., Reichenbecher, W., Steinbrecher, R. A., & Waßmann, F. (2019) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2019.00031 · Cited by 106 · see citing works
Young, J. K., Zastrow‐Hayes, G., Deschamps, S., Svitashev, S. K., Zaremba, M., Acharya, A., Paulraj, S., Peterson-Burch, B. D., Schwartz, C. S., Djukanovic, V., et al. (2019) · Scientific Reports · doi:10.1038/s41598-019-43141-6 · Cited by 103 · see citing works
Self-citationDefining CRISPR–Cas9 genome-wide nuclease activities with CIRCLE-seq
Lazzarotto, C. R., Nguyen, N. T., Tang, X., Malagon-Lopez, J., Guo, J. A., Aryee, M. J., Joung, J. K., & Tsai, S. Q. (2018) · Nature Protocols · doi:10.1038/s41596-018-0055-0 · Cited by 96 · see citing works
Pazi, K., Chathoth, K. T., Taylor, G. C. A., Meynert, A. M., Ballinger, T. J., Kelder, M. J. E., Lalevée, S., Sanli, I., Feil, R., & Wood, A. J. (2018) · PLoS Biology · doi:10.1371/journal.pbio.2005595 · Cited by 99 · see citing works
Kimberland, M. L., Hou, W., Alfonso-Pecchio, A. R., Wilson, S. P., Rao, Y., Zhang, S., & Lu, Q. (2018) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2018.08.007 · Cited by 96 · see citing works
Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges
Kalter, N., Fuster‐García, C., Silva, A., Ronco-Díaz, V., Roncelli, S., Turchiano, G., Gorodkin, J., Cathomen, T., Benabdellah, K., Lee, C. M., et al. (2025) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2025.102636 · Cited by 107 · see citing works
Epigenome engineering: new technologies for precision medicine
Sgro, A., & Blancafort, P. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa1000 · Cited by 96 · see citing works
CRISPR off-target detection with DISCOVER-seq
Wienert, B., Wyman, S. K., Yeh, C. D., Conklin, B. R., & Corn, J. E. (2020) · Nature Protocols · doi:10.1038/s41596-020-0309-5 · Cited by 91 · see citing works
Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pacesa, M., et al. (2024) · doi:10.1101/2024.04.22.590591 · Cited by 90 · see citing works
Self-citationSalk, J. J., & Kennedy, S. R. (2019) · Environmental and Molecular Mutagenesis · doi:10.1002/em.22342 · Cited by 89 · see citing works
Wang, Y., Liu, K. I., Sutrisnoh, N.-A. B., Srinivasan, H., Zhang, J., Li, J., Zhang, F., Richard, J. L. C., Xing, H., Shanmugam, R., et al. (2018) · Genome biology · doi:10.1186/s13059-018-1445-x · Cited by 87 · see citing works
Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites
Tao, J., Wang, Q., Mendez‐Dorantes, C., Burns, K. H., & Chiarle, R. (2022) · Nature Communications · doi:10.1038/s41467-022-31322-3 · Cited by 88 · see citing works
Agapito-Tenfen, S. Z., Okoli, A. S., Bernstein, M. J., Wikmark, O.-G., & Myhr, A. I. (2018) · Frontiers in Plant Science · doi:10.3389/fpls.2018.01874 · Cited by 86 · see citing works
Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments
Canver, M. C., Haeussler, M., Bauer, D. E., Orkin, S. H., Sanjana, N. E., Shalem, O., Yuan, G., Zhang, F., Concordet, J., & Pinello, L. (2018) · Nature Protocols · doi:10.1038/nprot.2018.005 · Cited by 87 · see citing works
Lopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82 · see citing works
Regulatory Status of Genome-Edited Organisms Under the Japanese Cartagena Act
Tsuda, M., Watanabe, K., & Ohsawa, R. (2019) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2019.00387 · Cited by 81 · see citing works
Improvements in Gene Editing Technology Boost Its Applications in Livestock
Périssé, I. V., Fan, Z., Singina, G. N., White, K. L., & Polejaeva, I. A. (2021) · Frontiers in Genetics · doi:10.3389/fgene.2020.614688 · Cited by 91 · see citing works
Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag
Liang, S., Liu, P., Ponnienselvan, K., Suresh, S., Chen, Z., Kramme, C. C., Chatterjee, P., Zhu, L. J., Sontheimer, E. J., Xue, W., et al. (2023) · Nature Methods · doi:10.1038/s41592-023-01859-2 · Cited by 79 · see citing works
Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture
Kawall, K., Cotter-Howells, J. D., & Then, C. (2020) · Environmental Sciences Europe · doi:10.1186/s12302-020-00361-2 · Cited by 77 · see citing works
Atkins, A., Chung, C.-H., Allen, A., Dampier, W., Gurrola, T. E., Sariyer, I. K., Nonnemacher, M., & Wigdahl, B. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.673022 · Cited by 66 · see citing works
Panel), E. P. O. G. M. O. (. G., Naegeli, H., Bresson, J., Dalmay, T., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejátko, J., Moreno, F. J., et al. (2020) · EFSA Journal · doi:10.2903/j.efsa.2020.6299 · Cited by 71 · see citing works
Ling, X., Chang, L., Chen, H., Gao, X., Yin, J., Zuo, Y., Huang, Y., Zhang, B., Hu, J., & Liu, T. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.09.021 · Cited by 71 · see citing works
Shapiro, J., Iancu, O., Jacobi, A. M., McNeill, M., Turk, R., Rettig, G. R., Amit, I., Tovin, A., Zohar Yakhini, B., Behlke, M. A., et al. (2020) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2020.04.027 · Cited by 69 · see citing works
Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy
Ferrari, S., Valeri, E., Conti, A., Scala, S., Aprile, A., Micco, R. D., Kajaste‐Rudnitski, A., Montini, E., Ferrari, G., Aiuti, A., et al. (2023) · Cell stem cell · doi:10.1016/j.stem.2023.04.014 · Cited by 71 · see citing works
Donohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.07.035 · Cited by 69 · see citing works
Self-citationHasanzadeh, A., Hamblin, M. R., Kiani, J., Noori, H., Hardie, J. M., Karimi, M., & Shafiee, H. (2022) · Nano Today · doi:10.1016/j.nantod.2022.101665 · Cited by 69 · see citing works
Guidelines for Optimized Gene Knockout Using CRISPR/Cas9
Campenhout, C. V., Cabochette, P., Veillard, A.-C., Laczik, M., Zelisko-Schmidt, A., Sabatel, C., Dhainaut, M., Vanhollebeke, B., Gueydan, C., & Kruys, V. (2019) · BioTechniques · doi:10.2144/btn-2018-0187 · Cited by 67 · see citing works
Evolving AAV-delivered therapeutics towards ultimate cures
He, X., Urip, B. A., Zhang, Z., Ngan, C. C., & Feng, B. (2021) · Journal of Molecular Medicine · doi:10.1007/s00109-020-02034-2 · Cited by 67 · see citing works
Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing
Li, J., Hong, S., Chen, W., Zuo, E., & Yang, H. (2019) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2019.11.002 · Cited by 63 · see citing works
Canaj, H., Hussmann, J. A., Li, H., Beckman, K. A., Goodrich, L., Cho, N. H., Li, Y. J., Santos, D. A., McGeever, A., Stewart, E. M., et al. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/841098 · Cited by 62 · see citing works
Marchetti, F., Cardoso, R., Chen, C. L., Douglas, G. R., Elloway, J. M., Escobar, P. A., Harper, T. A., Heflich, R. H., Kidd, D., Lynch, A. M., et al. (2023) · Mutation Research/Reviews in Mutation Research · doi:10.1016/j.mrrev.2023.108466 · Cited by 65 · see citing works
Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
Höijer, I., Johansson, J., Gudmundsson, S., Chin, C.-S., Bunikis, I., Häggqvist, S., Emmanouilidou, A., Wilbe, M., den Hoed, M., Bondeson, M.-L., et al. (2020) · Genome biology · doi:10.1186/s13059-020-02206-w · Cited by 64 · see citing works
Lea, R. A., & Niakan, K. K. (2019) · Nature Cell Biology · doi:10.1038/s41556-019-0424-0 · Cited by 62 · see citing works
Gene Editing and Genotoxicity: Targeting the Off-Targets
Blattner, G., Cavazza, A., Thrasher, A. J., & Turchiano, G. (2020) · Frontiers in Genome Editing · doi:10.3389/fgeed.2020.613252 · Cited by 62 · see citing works
ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected
Boutin, J., Cappellen, D., Rosier, J., Amintas, S., Dabernat, S., Bedel, A., & Moreau‐Gaudry, F. (2022) · The CRISPR Journal · doi:10.1089/crispr.2021.0120 · Cited by 57 · see citing works
CRISPR nuclease off-target activity and mitigation strategies
Wienert, B., & Cromer, M. K. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.1050507 · Cited by 62 · see citing works
Time to evolve: predicting engineered T cell-associated toxicity with next-generation models
Donnadieu, E., Luu, M., Alb, M., Anliker, B., Arcangeli, S., Bonini, C., Angelis, B. D., Choudhary, R., Espie, D., Galy, A., et al. (2022) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2021-003486 · Cited by 50 · see citing works
CRISPR system: Discovery, development and off-target detection
Chen, S., Yao, Y., Zhang, Y., & Fan, G. (2020) · Cellular Signalling · doi:10.1016/j.cellsig.2020.109577 · Cited by 61 · see citing works
Inhibition of HBV Expression in HBV Transgenic Mice Using AAV-Delivered CRISPR-SaCas9
Li, H., Sheng, C., Liu, H., Wang, S., Zhao, J., Yang, L., Jia, L., Li, P., Wang, L., Xie, J., et al. (2018) · Frontiers in Immunology · doi:10.3389/fimmu.2018.02080 · Cited by 60 · see citing works
Amini, L., Wagner, D. L., Rössler, U., Zarrinrad, G., Wagner, L. F., Vollmer, T., Wendering, D. J., Kornak, U., Volk, H., Reinke, P., et al. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.09.011 · Cited by 56 · see citing works
Dewari, P. S., Southgate, B., McCarten, K., Monogarov, G., O’Duibhir, E., Quinn, N., Tyrer, A., Leitner, M.-C., Plumb, C., Kalantzaki, M., et al. (2018) · eLife · doi:10.7554/elife.35069 · Cited by 58 · see citing works
Boyle, E. A., Becker, W. R., Bai, H., Chen, J. S., Doudna, J. A., & Greenleaf, W. J. (2021) · Science Advances · doi:10.1126/sciadv.abe5496 · Cited by 59 · see citing works
Wang, J., Zhang, C., & Feng, B. (2020) · Journal of Cellular and Molecular Medicine · doi:10.1111/jcmm.15039 · Cited by 60 · see citing works
Rose, J. C., Popp, N. A., Richardson, C. D., Stephany, J. J., Mathieu, J., Wei, C. T., Corn, J. E., Maly, D. J., & Fowler, D. M. (2020) · Nature Communications · doi:10.1038/s41467-020-16542-9 · Cited by 56 · see citing works
Tracking-seq reveals the heterogeneity of off-target effects in CRISPR–Cas9-mediated genome editing
Zhu, M., Xu, R., Yuan, J., Wang, J., Ren, X., Cong, T., You, Y., Ju, A., Xu, L., Wang, H., et al. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02307-y · Cited by 60 · see citing works
Breton, C. A., Clark, P., Wang, L., Greig, J. A., & Wilson, J. M. (2020) · BMC Genomics · doi:10.1186/s12864-020-6655-4 · Cited by 57 · see citing works
- Authors
- Cameron, P., Fuller, C. K., Donohoue, P. D., Jones, B. N., Thompson, M. S., Carter, M. M., Gradia, S., Vidal, B., Garner, E., Slorach, E. M., Lau, E., Banh, L. M., Lied, A. M., Edwards, L. S., Settle, A. H., Capurso, D., Llaca, V., Deschamps, S., Cigan, M., Young, J. K., & May, A. P.
- Affiliations
- Caribou Biosciences (United States) (US)
- Pioneer Hi-Bred (US)
- Corresponding author
- Young, Joshua K
- May, Andrew P
- ORCID
- Venue
- Nature Methods
- Published
- 2017-05-01
- Volume
- 14
- Issue
- 6
- Pages
- 600-606
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2611134270
- Cited by
- 443
- References
- 34
- Citations by year
- 2026: 34 · 2025: 59 · 2024: 51 · 2023: 45 · 2022: 57 · 2021: 40 · 2020: 64 · 2019: 39 · 2018: 36 · 2017: 17
- FWCI
- 16.98
- Type
- journal article
- Language
- en
- Update notices
- correction (2023-11-09)
- Open access
- closed
- Licence
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- 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, off-target mutations, single guide RNA, genome editing, nuclease activity
- MeSH terms
- Chromosome Mapping, Genome, Sequence Analysis, DNA, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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
