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Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
Open accessAbstract
CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.
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“Epigenetic editing with CRISPR/dCas9 is not without its limitations, as it can involve off-target effects [155, 159].”
From Cell-type-specific epigenetic effects of early life stress on the brain (2022) · cites this paper as [159]
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“It has been shown that more than three mismatches between the target sequence and the 20-nucleotide sgRNA can result in off-target effects.64”
From Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing (2021) · cites this paper as [64]
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Khare, P., Mandal, S., Verma, A. K., Parashar, D., Jadhav, K., & Gupta, S. K. (2024) · Elsevier eBooks · doi:10.1016/b978-0-323-91808-4.00006-7 · Cited by 2
Biosafety and biosecurity consideration in CRISPR-Cas
Hassan, N., Ali, H., Hassan, F., Ali, H., & Tawab, A. (2024) · Elsevier eBooks · doi:10.1016/b978-0-443-13229-2.00018-1 · Cited by 2
Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells
Kim, S., Matsushita, Y., Katagiri, T., & Maseda, H. (2025) · Scientific Reports · doi:10.1038/s41598-025-94071-5 · Cited by 2
Lv, X., Wei, Q., Zhi, Q., Liu, X., Fan, L., Niu, Y., Sun, H., Jin, K., Chen, G. H., Li, B., et al. (2025) · Journal of Animal Science · doi:10.1093/jas/skaf315 · Cited by 2
Rahimi, M., Schreiber, M., Habermann, H., Haake, L. R., Miskel, D., Tesfaye, D., Held-Hoelker, E., Brenig, B., Benedetti, C., & Hoelker, M. (2025) · Theriogenology · doi:10.1016/j.theriogenology.2025.117703 · Cited by 2
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CRISPR-Cas system: off-target effects and its possible solutions
Hao, Y. (2024) · International Conference on Biological Engineering and Medical Science (ICBioMed) · doi:10.1117/12.3012920 · Cited by 2
Wakatabi, K., Selvaraj, M. G., Guzmán-Prada, D. A., Cuásquer, J., López‐López, K., Endo, M., & Ishitani, M. (2023) · Revista Colombiana de Ciencias Hortícolas · doi:10.17584/rcch.2023v17i3.16120 · Cited by 2
Niu, J., Ram, A. F. J., & Punt, P. J. (2020) · Grand challenges in biology and biotechnology · doi:10.1007/978-3-030-29541-7_8 · Cited by 2
Reengineering of the CRISPR/Cas System
Khan, Z., Sattar, T., Siddique, M., Ali, Z., Khan, A. A., Aftab, S. O., Ghouri, M. Z., Sultan, Q., Gulzar, N., & Ahmad, F. (2022) · Springer eBooks · doi:10.1007/978-981-16-6305-5_5 · Cited by 2
Elacqua, J. J., Ranu, N., Dilorio, S. E., & Blainey, P. C. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/867937 · Cited by 2
A slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9Preprint
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Metadata
- Authors
- Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H.
- Affiliations
- McGovern Institute for Brain Research (US)
- ORCID
- Tee, Louis Y
- Venue
- Molecular Therapy - Nucleic Acids
- Publisher
- Elsevier BV
- Published
- 2015
- Volume
- 4
- Pages
- e264
- ISSN
- 2162-2531 (ISSN-L)
- PMC
- PMC4877446
- OpenAlex
- W2173737475
- Cited by
- 1,273
- References
- 82
- Citations by year
- 2026: 67 · 2025: 145 · 2024: 166 · 2023: 151 · 2022: 164 · 2021: 198 · 2020: 132 · 2019: 109 · 2018: 80 · 2017: 35 · 2016: 25 · 2015: 1
- FWCI
- 4.55
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-sa)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
- PubMed Central (publishedVersion, cc-by-nc-sa)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- 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 effects, genome editing, RNA-guided endonuclease, genome editing specificity, therapeutic genome editing
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