Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing
Open accessAbstract
To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a “core” sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of ‘super’ off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.
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In rhesus macaques, CAR T cells underwent a 4-day expansion in a G-Rex system after a single rapid transduction step, shortening manufacturing to 9 days.
“In another study, rhesus macaque CAR T cells underwent a 4-day expansion in a G-Rex system after a single rapid transduction step, shortening manufacturing to 9 days (115).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [115]
The Results section describes CRISPR/Cpf1 specificity assays in HEK293 and HeLa cells, not CAR T-cell manufacturing. Searches for rhesus macaques, G-Rex expansion, rapid transduction, and a 9-day manufacturing schedule found no passage addressing the claim in our copy. (LLM, conf=0.78)
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- Authors
- Wang, Y., Wang, M., Zheng, T., Hou, Y., Zhang, P., Tang, T., Wei, J., & Du, Q.
- Affiliations
- Peking University (CN)
- Chinese University of Hong Kong (HK)
- Nantong University (CN)
- Corresponding author
- Du, Quan
- Venue
- Scientific Reports
- Published
- 2020-02-10
- Volume
- 10
- Issue
- 1
- Pages
- 2269-2269
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC7010781
- OpenAlex
- W3004567324
- Cited by
- 31
- References
- 22
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 3 · 2023: 3 · 2022: 5 · 2021: 9 · 2020: 6
- FWCI
- 1.87
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Ministry of Science and Technology of the People's Republic of China (2014AA021103)
- National Natural Science Foundation of China (31571403)
- Natural Science Foundation of Beijing Municipality (2171001)
- 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 › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas12a, CRISPR/Cas9, editing specificity, target-site mutations, point mutations, core sequence, single guide RNA
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Bacterial Proteins, Endodeoxyribonucleases, Humans, Mutation, Nucleotides, RNA, Guide, Kinetoplastida, HEK293 Cells, CRISPR-Cas Systems, CRISPR-Associated Proteins
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