Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil‐DNA glycosylase
Abstract
Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.
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Cytosine base editing-which achieves precise C-to-T substitutions without double-strand breaks, donor DNA, or Cas nuclease toxicity-has been demonstrated in Synechococcus elongatus and subsequently extended to Synechocystis and Anabaena with multiplex capability and integrated counter-selection for plasmid curing.
“Complementing these approaches, cytosine base editing—which achieves precise C-to-T substitutions without double-strand breaks, donor DNA, or Cas nuclease toxicity—has been demonstrated in Synechococcus elongatus [124] and subsequently extended to Synechocystis and Anabaena with multiplex capability and integrated counter-selection for plasmid curing [125].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [125]
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- Authors
- Lee, M., Heo, Y. B., & Woo, H. M.
- Affiliations
- Sungkyunkwan University (KR)
- Corresponding author
- Woo, Han Min
- Venue
- The Plant Journal
- Publisher
- Wiley
- Published
- 2023-01-11
- Volume
- 113
- Issue
- 3
- Pages
- 610-625
- ISSN
- 0960-7412 (ISSN-L)
- 1365-313X
- OpenAlex
- W4312128889
- Cited by
- 15
- References
- 61
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 5 · 2023: 3
- FWCI
- 0.90
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Plant Journal (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Research Foundation of Korea (2022M3A9F3082328, 2022M3J5A1056117, 2022M3J5A1062990, 2022R1A2C2008242, 2022R1A4A5032720)
- 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 › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- cytosine base editing, uracil-DNA glycosylase, gene repression, cyanobacteria, cytidine deaminase, archaea, green algae
- MeSH terms
- Gene Editing, Cyanobacteria, Cytosine, DNA, DNA Repair, Uracil-DNA Glycosidase
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