数字农科院2.0

Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice

文献类型： 外文期刊

作者： Hu, Jianjian;Li, Xue;Gao, Yuhong;Guo, Yifan;Liu, Yini;Wang, Chen;Xu, Gencheng;Du, Chaoyue;Liu, Shijia;Zhao, Zhigang;Wang, Yihua;Wu, Yufeng;Dong, Xiaoou;Li, Chao;Wan, Jianmin

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关键词： adenine base editor;CH-ABE;high-fidelity;SpRY;TadA8e

期刊名称： PLANT BIOTECHNOLOGY JOURNAL

ISSN： 1467-7644

年卷期： 2025 年

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收录情况： SCIE（2025版）

摘要： Adenine base editors (ABEs) produce precise A-to-G conversion in the genomic target sites without causing double-strand breaks. However, the hyperactive adenosine deaminase TadA8e raises safety concerns on genome-wide off-target edits. We engineered 11 chimeric proteins for ABEs (CP-ABEs) by embedding hyperactive TadA8e within Cas9 nickase to minimise the sgRNA-independent off-target effects. Four CP-ABEs exhibited robust on-target activity with minimal sgRNA-independent off-target edits. Then we developed four chimeric high-fidelity ABEs (CH-ABEs) to minimise both sgRNA-dependent and sgRNA-independent off-target effects by employing high-fidelity Cas9 variants. The CH-ABEs achieved reductions of up to 7.0-fold and 79.4-fold in the respective off-target edits, while generating 22.0%-72.4% homozygous and biallelic rice mutants. Whole-genome and whole-transcriptome sequencing (WGS/WTS) confirmed the specificity of CH-ABEs. Incorporating Sniper2L into CH-ABEs further enhanced both specificity and on-target activity. Two PAM-less SpRY variants (SpRY-K2, SpRY-KK) expanded the targeting scope of CP-ABEs and boosted activity by 80.0%. Furthermore, we demonstrated that CP-ABE8e-RYKK could discriminate paralogous targets in rice and successfully applied it to create herbicide-resistant rice by precisely installing the OsALS-K591E mutation.

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