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Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants
Open accessAbstract
CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity . In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity . Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.
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“Some studies suggest that higher GC content in the gRNA can stabilize the DNA-RNA duplex, potentially increasing on-target activity”
From Hybrid Neural Networks for Off-Target Prediction in Gene Editing: Integrating Sequence and Biophysical Features (2025) · cites this paper as [1]
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“while others indicate that target sequences with a GC content higher than 70% may increase the likelihood of off-target effects.”
From Hybrid Neural Networks for Off-Target Prediction in Gene Editing: Integrating Sequence and Biophysical Features (2025) · cites this paper as [1]
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Basic local alignment search tool
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990) · Journal of Molecular Biology · doi:10.1006/jmbi.1990.9999 · Cited by 98,669
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 · cited 4×
“It was considered that mismatches occurring within the first nucleotides proximal to the PAM determine the occurrence of off-target effects, leading to a decreased or even complete abolition of off-target effects (Hsu et al., 2013; Endo et al., 2015; Hahn and Nekrasov, 2018).”
BackgroundInfluentialHigh-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
Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014) · Nature Biotechnology · doi:10.1038/nbt.2969 · Cited by 2,072
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · cited 1×
“These results are consistent with previous findings (e.g., Mali et al., 2013; Endo et al., 2015; Jiang et al., 2015).”
ResultCHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing
Montague, T. G., Cruz, J. M. B. S., Gagnon, J. A., Church, G. M., & Valen, E. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku410 · Cited by 1,246
DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins
Woo, J. W., Kim, J., Kwon, S. I., Corvalán, C., Cho, S. W., Kim, H., Kim, S.-G., Kim, S.-T., Choe, S., & Kim, J.-S. (2015) · Nature Biotechnology · doi:10.1038/nbt.3389 · Cited by 1,219
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Chen, J. S., Dagdas, Y. S., Kleinstiver, B. P., Welch, M. M., Sousa, A. A., Harrington, L. B., Sternberg, S. H., Joung, J. K., Yildiz, A., & Doudna, J. A. (2017) · Nature · doi:10.1038/nature24268 · Cited by 1,168
The Rate and Molecular Spectrum of Spontaneous Mutations in Arabidopsis thaliana
Ossowski, S., Schneeberger, K., Lucas‐Lledó, J. I., Warthmann, N., Clark, R. M., Shaw, R. G., Weigel, D., & Lynch, M. (2009) · Science · doi:10.1126/science.1180677 · Cited by 1,166
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
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Research works printed but not linked: 3
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- 2018, JMP® 14 Fitting Linear Models, Chu ban:
- Binary logistic regression – what, when, and how, WalshS. JMP Discovery Conference 20162016
Cited by
Sorted by citations
Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants
Son, S., & Park, S. R. (2022) · Frontiers in Plant Science · doi:10.3389/fpls.2022.902413 · Cited by 113
Genome editing around the globe: An update on policies and perceptions
Sprink, T., Wilhelm, R., & Hartung, F. (2022) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiac359 · Cited by 105
Self-citationOccurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants
Sturme, M. H. J., van der Berg, J. P., Bouwman, L. M. S., de Schrijver, A., de Maagd, R. A., Kleter, G. A., & Battaglia, E. (2022) · ACS Agricultural Science & Technology · doi:10.1021/acsagscitech.1c00270 · Cited by 99
Enhancement of specialized metabolites using CRISPR/Cas gene editing technology in medicinal plants
Das, S., Kwon, M., & Kim, J. (2024) · Frontiers in Plant Science · doi:10.3389/fpls.2024.1279738 · Cited by 82
The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model
Li, Y., Xu, J., Guo, X., Li, Z., Cao, L., Liu, S., Guo, Y. Q., Wang, G., Luo, Y., Zhang, Z., et al. (2023) · Genome biology · doi:10.1186/s13059-023-02860-w · Cited by 84
Metadata
- Authors
- Modrzejewski, D., Hartung, F., Lehnert, H., Sprink, T., Kohl, C., Keilwagen, J., & Wilhelm, R.
- Affiliations
- Julius Kühn-Institut (DE)
- Corresponding author
- Modrzejewski, Dominik
- Hartung, Frank
- ORCID
- Venue
- Frontiers in Plant Science
- Publisher
- Frontiers Media SA
- Published
- 2020-11-23
- Volume
- 11
- Pages
- 574959-574959
- ISSN
- 1664-462X (ISSN-L)
- PMC
- PMC7719684
- OpenAlex
- W3106735065
- Cited by
- 151
- References
- 63
- Citations by year
- 2026: 22 · 2025: 25 · 2024: 27 · 2023: 30 · 2022: 32 · 2021: 14
- FWCI
- 1.96
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Plant Science (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- OpenAgrar (submittedVersion, cc-by)
- Funders
- Bundesministerium für Bildung und Forschung
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- CRISPR-Cas, off-target effects, genome editing, plants, GC content, nuclease variant, SpCas9, PAM, delivery methods
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