Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases
Open accessAbstract
The development of customizable sequence-specific nucleases such as TALENs, ZFNs and the powerful CRISPR/Cas9 system has revolutionized the field of genome editing. The CRISPR/Cas9 system is particularly versatile and has been applied in numerous species representing all branches of life. Regardless of the target organism, all researchers using sequence-specific nucleases face similar challenges: confirmation of the desired on-target mutation and the detection of off-target events. Here, we evaluate the most widely-used methods for the detection of on-target and off-target mutations in terms of workflow, sensitivity, strengths and weaknesses.
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LNP delivery in vivo eliminates off-target effects.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [103]
Section 3.1 (page unavailable) says delivery method can affect off-target frequency: transient mRNA delivery or Cas9 protein and gRNA produced in vitro showed lower off-target activity than plasmid expression. It does not identify LNP delivery, establish an in vivo result, or say that this delivery eliminates off-target effects. (LLM, conf=0.84)
Experimental — accuracy not yet measuredAAV delivery in vivo may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [103]
Section 3.1 says delivery method affects off-target mutation frequency and reports lower off-target activity with transient Cas9 delivery than with plasmid expression. It does not address AAV delivery in vivo or say that off-target mutations accumulate over time. (LLM, conf=0.80)
Experimental — accuracy not yet measuredWhen indels are deposited at coding sequences of a given gene, NHEJ can cause frameshift mutations, resulting in non-sense-mediated mRNA decay and gene silencing.
“When the indels are deposited at the coding sequences of a given gene, NHEJ can cause frameshift mutations, resulting in non-sense-mediated mRNA decay and gene silencing (Wang et al., 2016a; Zischewski et al., 2017; Lindeboom et al., 2019).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Zischewski et al., 2017
LNP delivery in vivo can be quickly degraded.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [103]
Section 3.1 says that a Cas9–gRNA ribonucleoprotein is subject to endogenous degradation, but it does not address degradation of lipid nanoparticles (LNPs) in vivo. Searches for LNP delivery and in-vivo degradation found no passage supporting that specific claim; no page number was available in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredAAV delivery in vivo lasts for years in terminally differentiated cell types.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [103]
Section 3.1 discusses how transient mRNA or protein delivery affects off-target activity, but does not address AAV persistence in vivo. Searches for AAV, terminally differentiated cells, and delivery lasting for years found no passage supporting the claim in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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“When the indels are deposited at the coding sequences of a given gene, NHEJ can cause frameshift mutations, resulting in nonsense-mediated mRNA decay and gene silencing (Wang et al., 2016a; Zischewski et al., 2017; Lindeboom et al., 2019).”
BackgroundCRISPR/Cas system: recent advances and future prospects for genome editing
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Aubi, O., Prestegård, K. S., Jung‐KC, K., Shi, T.-J. S., Ying, M., Grindheim, A. K., Scherer, T., Ulvik, A., McCann, A., Spriet, E., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-22107-1 · Cited by 29 · see citing works
Biomaterial-Based CRISPR/Cas9 Delivery Systems for Tumor Treatment
Li, M., Chen, F., Yang, Q., Tang, Q., Xiao, Z., Tong, X., Zhang, Y., Lei, L., & Li, S. (2024) · Biomaterials Research · doi:10.34133/bmr.0023 · Cited by 31 · see citing works
CRISPR-Mediated Modification of DNA Methylation Pattern in the New Era of cancer therapy
Maroufi, F., Maali, A., Abdollahpour‐Alitappeh, M., Ahmadi, M. H., & Azad, M. (2020) · Epigenomics · doi:10.2217/epi-2020-0110 · Cited by 29 · see citing works
Dashnau, J. L., Xue, Q., Nelson, M., Law, E., Cao, L., & Hei, D. J. (2022) · Cytotherapy · doi:10.1016/j.jcyt.2022.08.001 · Cited by 30 · see citing works
- Authors
- Zischewski, J., Fischer, R., & Bortesi, L.
- Affiliations
- RWTH Aachen University (DE)
- Fraunhofer Institute for Molecular Biology and Applied Ecology (DE)
- Corresponding author
- Bortesi, Luisa
- Venue
- Biotechnology Advances
- Publisher
- Elsevier BV
- Published
- 2017-01
- Volume
- 35
- Issue
- 1
- Pages
- 95-104
- ISSN
- 0734-9750 (ISSN-L)
- OpenAlex
- W2565555627
- Cited by
- 323
- References
- 78
- Citations by year
- 2026: 14 · 2025: 29 · 2024: 32 · 2023: 31 · 2022: 35 · 2021: 41 · 2020: 40 · 2019: 46 · 2018: 37 · 2017: 18
- FWCI
- 3.04
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Biotechnology Advances (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Research Publications (Maastricht University) (publishedVersion)
- RWTH Publications (RWTH Aachen) (publishedVersion, other-oa)
- Fraunhofer-Publica (Fraunhofer-Gesellschaft) (submittedVersion)
- Funders
- European Research Council (269110)
- 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 › DNA and Nucleic Acid Chemistry
- Keywords
- CRISPR/Cas9, off-target mutations, TALEN, zinc finger nucleases, sequence-specific nucleases, genome editing, sensitivity
- MeSH terms
- Gene Editing, Animals, Base Sequence, Cell Line, Deoxyribonucleases, Humans, Mutation, Mice, CRISPR-Cas Systems
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