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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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“| 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]
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In the citing paper
“| 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]
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“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
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“| 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]
Experimental — accuracy not yet measuredThe check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“| 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]
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Paving the way towards precise and safe CRISPR genome editing
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Detection of genome edits in plants—from editing to seed
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Metadata
- 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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