Massively targeted evaluation of therapeutic CRISPR off-targets in cells
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Abstract
Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.
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CIRCLE-seq can detect genome-wide off-target sites without performing WGS.
“CIRCLE-seq is another method that can detect genome-wide off-target sites without performing WGS.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Pan et al., 2022
The References identify CIRCLE-seq as a genome-wide CRISPR-Cas9 off-target screen, and the Results describe it detecting off-target sites through cleavage of cell-free DNA. Our copy does not state whether CIRCLE-seq avoids whole-genome sequencing, so it does not establish that part of the claim. (LLM, conf=0.80)
Experimental — accuracy not yet measuredA primary limitation of GUIDE-seq is the low delivery efficiency of dsODNs into cells, resulting in detection of only 30%-50% of all DSBs.
“A primary limitation of GUIDE-seq is relevant to the low delivery efficiency of dsODNs into cells, which results in detection of only 30%–50% of all the DSBs (Tsai et al., 2015; Pan et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Pan et al., 2022
The Introduction (page unavailable) says GUIDE-seq relies on inserting double-stranded DNA at double-strand breaks, and the Results section on SURRO-seq validation likewise describes insertion of a double-stranded oligonucleotide. Neither passage says that delivery into cells is inefficient or that GUIDE-seq detects only 30%–50% of DSBs; searches for those terms found no such estimate in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measured
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Massively Targeted Evaluation of Therapeutic CRISPR Off-Targets in CellsPreprint
Pan, X., Qu, K., Yuan, H., Xiang, X., Anthon, C., Pashkova, L., Liang, X., Han, P., Corsi, G. I., Xu, F., et al. (2022) · Research Square · doi:10.21203/rs.3.rs-1427273/v1 · Cited by 3 · see citing works
Self-citation
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1 of the 79 references couldn’t be matched to a known paper.
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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
“A primary limitation of GUIDE-seq is relevant to the low delivery efficiency of dsODNs into cells, which results in detection of only 30%–50% of all the DSBs (Tsai et al., 2015; Pan et al., 2022).”
BackgroundAssessing 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
Chemically Modified Platforms for Better RNA Therapeutics
Shi, Y., Zhen, X., Zhang, Y., Li, Y., Koo, S., Saiding, Q., Kong, N., Liu, G., Chen, W., & Tao, W. (2024) · Chemical Reviews · doi:10.1021/acs.chemrev.3c00611 · Cited by 184 · 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
Self-citationLopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82 · see citing works
Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints
Chen, Q., Chuai, G., Zhang, H., Tang, J., Duan, L., Guan, H., Li, W., Li, W., Wen, J., Zuo, E., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-42695-4 · Cited by 82 · see citing works
CRISPR delivery with extracellular vesicles: Promises and challenges
Berggreen, A. H., Petersen, J. L., Lin, L., Benabdellah, K., & Luo, Y. (2023) · Journal of Extracellular Biology · doi:10.1002/jex2.111 · Cited by 45 · see citing works
Self-citationExosome-mediated delivery of CRISPR-Cas9: A revolutionary approach to cancer gene editing
Balaraman, A. K., Babu, M. A., Moglad, E. H. O., Mandaliya, V. B., Rekha, M. M., Gupta, S., Prasad, G. V. S., Kumari, M., Chauhan, A. S., Ali, H., et al. (2024) · Pathology - Research and Practice · doi:10.1016/j.prp.2024.155785 · Cited by 44 · see citing works
Adeno-Associated Virus (AAV) - Based Gene Therapies for Retinal Diseases: Where are We?
Ail, D., Malki, H., Zin, E. A., & Dalkara, D. (2023) · The Application of Clinical Genetics · doi:10.2147/tacg.s383453 · Cited by 40 · see citing works
Madan, E., Palma, A. M., Vudatha, V., Kumar, A., Bhoopathi, P., Wilhelm, J., Bernaś, T., Martin, P. C. N., Bilolikar, G., Gogna, A., et al. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02453-3 · Cited by 31 · see citing works
Self-citationA versatile CRISPR/Cas9 system off-target prediction tool using language model
Du, W., Zhao, L., Diao, K., Zheng, Y., Yang, Q., Zhu, Z., Zhu, X., & Tang, D. (2025) · Communications Biology · doi:10.1038/s42003-025-08275-6 · Cited by 32 · see citing works
Genome editing in maize and sorghum: A comprehensive review of CRISPR/Cas9 and emerging technologies
Namata, M. J., Xu, J., Habyarimana, E., Palakolanu, S. R., Wang, L., & Li, J. (2025) · The Plant Genome · doi:10.1002/tpg2.70038 · Cited by 35 · see citing works
Fitness effects of CRISPR endonucleases in Drosophila melanogaster populations
Langmüller, A. M., Champer, J., Lapinska, S., Xie, L., Metzloff, M., Champer, S. E., Liu, J., Xu, Y., Du, J., Clark, A. G., et al. (2022) · eLife · doi:10.7554/elife.71809 · Cited by 23 · see citing works
Self-citationThe Interface of Gene Editing with Regenerative Medicine
Farag, V. E., Devey, E., & Leong, K. W. (2024) · Engineering · doi:10.1016/j.eng.2024.10.019 · Cited by 21 · see citing works
Xue, Y., Hu, M., Cha, S., Xue, C., & Dong, N. (2025) · Acta Biomaterialia · doi:10.1016/j.actbio.2025.10.012 · Cited by 12 · see citing works
Cavazza, A., Hendel, A., Bak, R. O., Rı́o, P., Güell, M., Lainšček, D., Arechavala‐Gomeza, V., Peng, L., Hapil, F. Z., Harvey, J. P., et al. (2023) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2023.102066 · Cited by 12 · see citing works
Self-citationMekonnen, A. M., Seong, K., Kim, H., & Park, J. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkaf389 · Cited by 11 · see citing works
Shining light on CRISPR/Cas9 therapeutics for inherited retinal diseases
Geiger, A., Kennedy, J. G., Staker, L. G., Wensel, T. G., Casson, R. J., & Thomas, P. Q. (2025) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2025.101376 · Cited by 11 · see citing works
Methods for detecting off-target effects of CRISPR/Cas9
Xu, Y., Zhou, S., Wang, L.-Y., Zhang, R., Li, K., Qian, Z., & Li, X. (2025) · Biotechnology Advances · doi:10.1016/j.biotechadv.2025.108750 · Cited by 11 · see citing works
Rahman, M. A., Akter, S., Ashrafudoulla, M., Jung, S.-J., Rapak, M. T., & Ha, S. (2025) · Food Research International · doi:10.1016/j.foodres.2025.117803 · Cited by 7 · see citing works
Sun, Y., Qu, K., Corsi, G. I., Anthon, C., Pan, X., Xiang, X., Jensen, L. J., Lin, L., Luo, Y., & Gorodkin, J. (2025) · Nature Communications · doi:10.1038/s41467-025-65200-5 · Cited by 7 · see citing works
Self-citationCRISPR technology in human diseases
Feng, Q., Li, Q., Zhou, H., Wang, Z., Lin, C., Jiang, Z., Liu, T., & Wang, D. (2024) · MedComm · doi:10.1002/mco2.672 · Cited by 7 · see citing works
Medea: An AI agent for therapeutic reasoning across biological contextsPreprint
Sui, P., Li, M. M., Munson, B. P., Gao, S., Shen, W., Giunchiglia, V., Shen, A. N., Huang, Y., Kong, Z., Licon, K., et al. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.01.16.696667 · Cited by 11 · see citing works
Zhao, X., Qu, K., Curci, B., Yang, H., Bolund, L., Lin, L., & Luo, Y. (2023) · Biomolecules · doi:10.3390/biom13050870 · Cited by 6 · see citing works
Self-citationNg, K. P., Seng, M. S.-F., & Leung, W. H. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101462 · Cited by 4 · see citing works
CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detectionPreprint
Labun, K., Rio, O., Tjeldnes, H., Świrski, M. I., Komisarczuk, A. Z., Haapaniemi, E. M., & Valen, E. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.01.06.603201 · Cited by 4 · see citing works
Ursch, L. T., Müschen, J. S., Ritter, J. M., Klermund, J., Bernard, B. E., Kolb, S., Warmuth, L., Andrieux, G., Miller, G., Jiménez‐Muñoz, M., et al. (2024) · Cell Reports Medicine · doi:10.1016/j.xcrm.2024.101846 · Cited by 3 · see citing works
Scalable assessment of genome editing off-targets associated with genetic variantsPreprint
Lin, J., Nguyen, M. A., Lin, L. Y., Zeng, J., Verma, A., Neri, N. R., da Silva, L. F., Mucci, A., Wolfe, S. A., Shaw, K. L., et al. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.07.24.605019 · Cited by 3 · see citing works
Self-citationCancer gene therapy: historical perspectives, current applications, and future directions
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Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos
Kim, J., Yoon, J. D., Chen, J., Lee, J., Lee, H. J., Whitworth, K. M., Redel, B. K., Prather, R. S., & Lee, K. (2026) · Journal of Animal Science · doi:10.1093/jas/skag030 · Cited by 2 · see citing works
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Chromatic grains: Revolutionizing nutrition with pigmented cereals and fortified foods
Ahmed, S. S., Kumar, J., GARG, N. K., & Gahlaut, V. (2025) · Journal of Food Measurement & Characterization · doi:10.1007/s11694-025-03681-0 · Cited by 1 · see citing works
World Sickle Cell Day 2022: Progress & prospects
Steinberg, M. H. (2022) · International Journal of Microbiology Research · doi:10.4103/ijmr.ijmr_1049_22
CRISPR: fundamental principles and implications for anaesthesia
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Navigating off-target effects in CRISPR-based genome editing for safer gene therapies
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CRISPR-Based Genome Editing and Delivery Systems: Advances, Challenges and Future Directions
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Nano Delivery Systems for In Vivo CRISPR/Cas-Based Gene Therapy: From Vectors to Clinical Outcomes
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- Authors
- Pan, X., Qu, K., Yuan, H., Xiang, X., Anthon, C., Pashkova, L., Liang, X., Han, P., Corsi, G. I., Xu, F., Liu, P., Zhong, J., Zhou, Y., Ma, T., Jiang, H., Liu, J., Wang, J., Jessen, N., Bolund, L., Yang, H., Xu, X., Church, G. M., Gorodkin, J., Lin, L., & Luo, Y.
- Affiliations
- BGI Group (China) (CN)
- University of Copenhagen (DK)
- Aarhus University (DK)
- Harvard University (US)
- Corresponding author
- Church, George M.
- Gorodkin, Jan
- Lin, Lin
- Luo, Yonglun
- ORCID
- Venue
- Nature Communications
- Published
- 2022-07-13
- Volume
- 13
- Issue
- 1
- Pages
- 4049-4049
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC9279339
- OpenAlex
- W4285088294
- Cited by
- 42
- References
- 79
- Citations by year
- 2026: 7 · 2025: 16 · 2024: 9 · 2023: 7 · 2022: 3
- FWCI
- 2.86
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- European Commission (HORIZON2020, 899417)
- Novo Nordisk
- Novo Nordisk Fonden (NNF17SA0031230-1, NNF21OC0071031, NNF21OC0068988, NNF21OC0072031)
- National Institutes of Health
- Sanming Project of Medicine in Shenzhen (2019B1515120034, SZSM201612074)
- National Human Genome Research Institute (RM1HG008525)
- Steno Diabetes Center Aarhus
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- RNA-guided nucleases, indels, wobble pairing, binding free energy, nuclease specificity, cancer genes, gene therapy safety
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cell Line, Endonucleases, Humans, Ribonucleases, 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.
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