CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity
Open accessAbstract
The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.
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In-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.
“Moreover, in-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [14]
The Results (page not provided) report off-target cleavage and repair outcomes including point mutations, insertions, and deletions, including gross chromosomal deletions. The Discussion (page not provided) suggests that translocations might occur, but does not report observing them; the claim overstates that outcome. (LLM, conf=0.90)
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- Authors
- Cradick, T. J., Fine, E. J., Antico, C. J., & Bao, G.
- Affiliations
- Georgia Institute of Technology (US)
- Corresponding author
- Bao, Gang
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2013-08-10
- Volume
- 41
- Issue
- 20
- Pages
- 9584-9592
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC3814385
- OpenAlex
- W2132401721
- Cited by
- 590
- References
- 31
- Citations by year
- 2026: 14 · 2025: 19 · 2024: 26 · 2023: 23 · 2022: 28 · 2021: 44 · 2020: 50 · 2019: 42 · 2018: 41 · 2017: 44 · 2016: 81 · 2015: 79 · 2014: 83 · 2013: 16
- FWCI
- 38.64
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- National Science Foundation (1148903)
- National Institutes of Health
- National Eye Institute (PN2EY018244)
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, off-target cleavage, beta-globin, CCR5, CCR2, protospacer adjacent motif, mismatch tolerance, chromosomal deletion, genome editing specificity
- MeSH terms
- Chromosome Deletion, Endodeoxyribonucleases, Humans, Receptors, CCR5, DNA Cleavage, beta-Globins, Genetic Loci, HEK293 Cells, CRISPR-Cas Systems, CRISPR-Associated Proteins
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