Detection of CRISPR/Cas9-Generated off-target effect by integration-defective lentiviral vector
Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.
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Viral transduction for Cas9/sgRNA delivery in cell culture may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in cell culture [89–94] | Viral transduction | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [89]
Plasmid transfection for Cas9/sgRNA delivery in cell culture may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in cell culture [89–94] | Plasmid transfection | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [89]
RNP electroporation for Cas9/sgRNA delivery in cell culture eliminates off-target effects.
“| Delivery methods improvement | Application in cell culture [89–94] | RNP electroporation: Rapid transfection and turnover | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [89]
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Off-target effects in CRISPR/Cas9 gene editing
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(2020) · Methods in molecular biology · doi:10.1007/978-1-0716-0687-2 · Cited by 3 · see citing works
- Authors
- Wang, X., Wu, Y., & Yee, J.-K.
- Affiliations
- Guangzhou Medical University (CN)
- City Of Hope National Medical Center (US)
- Corresponding author
- Yee, Jiing-Kuan
- Venue
- Methods in molecular biology
- Publisher
- Springer US
- Published
- 2020-09-15
- Volume
- 2162
- Pages
- 243-260
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W3084546431
- Cited by
- 5
- References
- 14
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 1 · 2021: 1 · 2020: 1
- FWCI
- 1.02
- Type
- book chapter
- Language
- en
- Open access
- closed
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, off-target effects, genome editing, deep sequencing, zinc finger nucleases, TALEN
- MeSH terms
- Gene Editing, Genetic Vectors, Humans, Lentivirus, Virus Integration, Gene Targeting, HEK293 Cells, CRISPR-Cas Systems
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