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Efficient C-to-G base editing in rice with reduced C-to-T byproducts using glycosylase-based cytosine base editors
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DOI：10.1016/j.cj.2025.06.021.png)
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Abstract
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Base editors are essential tools for precise genome editing in plants. However, achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging. In this study, we present the development and evaluation of a novel glycosylase-based cytosine base editor (gCBE) for efficient C-to-G editing in rice. Unlike traditional cytosine base editors, which rely on cytosine deamination, gCBE directly excises cytosine to generate an apurinic/apyrimidinic (AP) site, thus circumventing the deamination step and reducing the production of C-to-T byproducts. We constructed several gCBE variants, including N-gCBE, M-gCBE, and C-gCBE, by fusing engineered human UDG2 (UNG*) to SpCas9 nickase (nSpCas9, D10A) and tested their editing efficiency and specificity in rice. Our results demonstrate that M-gCBE achieved efficient C-to-G editing (6.3% to 37.5%) similar to OsCGBE (9.4% to 28.1%) at most targets, though with site-dependent variations. Notably, gCBE tools showed a marked reduction in C-to-T byproducts, with average C-to-T mutation rates of 12.5% for N-gCBE and 16.7% for M-gCBE, compared to 53.1% for OsCGBE. Notably, both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T0 generation, a key advantage over OsCGBE, which predominantly generated C-to-T mutations. Off-target analysis revealed minimal off-target effects with M-gCBE, highlighting its potential for high-precision genome editing. These findings suggest that gCBE tools, particularly M-gCBE, are highly efficient and precise, providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.
Keywords:
Base editing
C-to-G mutation
Rice
Glycosylase
Off-target effects
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