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Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors
Open accessAbstract
CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications 1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex 3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells 5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor 7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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“they create new formats of off-target effects such as RNA editing and sgRNA-independent DNA editing”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Grünewald et al., 2019
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“While these methods have been used in cultured cells, they are not yet ready for clinical use until matters of specificity 33,34 and delivery are addressed.”
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“The RNA off-target effects can be detected by RNA-seq”
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“Although CBE and ABE greatly reduce the classic off-target effects of CRISPR/Cas9 systems”
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“These systems induce transient conversions, potentially minimising undesired physiological changes compared to DNA editing. 90”
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Metadata
- Authors
- Grünewald, J., Zhou, R., Garcia, S. P., Iyer, S., Lareau, C. A., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Joung, J. Keith
- ORCID
- Venue
- Nature
- Published
- 2019-04-17
- Volume
- 569
- Issue
- 7756
- Pages
- 433-437
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC6657343
- OpenAlex
- W2939749480
- Cited by
- 663
- References
- 37
- Citations by year
- 2026: 66 · 2025: 74 · 2024: 80 · 2023: 100 · 2022: 84 · 2021: 106 · 2020: 104 · 2019: 48
- FWCI
- 28.72
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- National Human Genome Research Institute (RM1HG009490)
- National Institute of General Medical Sciences (R35GM118158)
- 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
- cytosine base editors, APOBEC1, RNA editing, off-target effects, adenine base editors, deaminase activity, human cells
- MeSH terms
- APOBEC-1 Deaminase, Gene Editing, Animals, Base Sequence, Cytosine, Deamination, Humans, Mutation, RNA, Substrate Specificity, RNA Editing, Rats, Hep G2 Cells, HEK293 Cells, Transcriptome, CRISPR-Cas Systems
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