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Engineering Adenine Deaminase TadA for Precise and PAM-Flexible Point Mutagenesis and Gradient-Tuning Endogenous Protein Design

文献类型： 外文期刊

作者： Sun, Kangli 1 ; Cheng, Si 3 ; Chai, Nan 2 ; Mi, Jianing 3 ; Zhang, Ruixiang 2 ; Qian, Qian 1 ; Zheng, Zhiye 2 ; Chen, Ke 1 ; Zeng, Dongchang 4 ; Peng, Xin 1 ; Shen, Mengyuan 1 ; Zhou, Degui 1 ; Zhu, Qinlong 2 ; Liu, Qi 1 ; Tan, Jiantao 1 ;

作者机构： 1.Guangdong Acad Agr Sci, Minist Agr & Rural Affairs, Rice Res Inst, Key Lab Genet & Breeding High Qual Rice Southern C, Guangzhou 510640, Peoples R China

2.South China Agr Univ, Coll Agr, Guangdong Basic Res Ctr Excellence Precise Breedin, State Key Lab Conservat & Utilizat Subtrop Agrobio, Guangzhou 510642, Peoples R China

3.Chinese Med Guangdong Lab, Zhuhai 529031, Peoples R China

4.Guangxi Normal Univ, Univ Engn Res Ctr Bioinformat & Genet Improvement, Guangxi Key Lab Landscape Resources Conservat & Su, Guilin 541006, Peoples R China

关键词： Badh2; gradient-tuned protein design; point mutagenesis; precise base editing; TadA variants

期刊名称：ADVANCED SCIENCE （ 影响因子：14.1； 五年影响因子：15.6 ）

ISSN：

年卷期： 2025 年

页码：

收录情况： SCI

摘要： Base editing enables precise nucleotide substitutions within a relatively broad editing window (5-6 nucleotides). However, considerable bystander editing significantly compromise its accuracy. Point mutagenesis, a powerful approach for gradient-tuning protein function, facilitates the generation of diverse plant phenotypes to meet the demands of complex environments and consumer preferences. Here, a series of plant base editors is engineered by fusing three optimized TadA8e variants, TadA9, TadA-LM, and TadA-dual, with a PAM-flexible SpRY nickase (SpRYn, with 5 '-NNN PAM recognition). These editors enable A-to-G, C-to-T, and dual-base (simultaneous A-to-G and C-to-T) conversions within a highly condensed active window (1-3 nucleotides). Performance evaluations reveal that the TadDBE (TadA Dual-Base Editor) achieves the most robust outcomes, delivering dual-base editing efficiencies ranging from 2.3% to 61.4%, while maintaining minimal off-target activity. Utilizing TadDBE, targeted point mutagenesis is performed on OsBadh2, a gene encoding betaine aldehyde dehydrogenase that plays a critical role in the biosynthesis of 2-acetyl-1-pyrroline (2-AP), a key aromatic compound. This approach yields rice lines exhibiting gradient-tuned aromatic profiles and optimized levels of 2-AP and gamma-aminobutyric acid (GABA). These evolved TadA-derived editors provide a precise, PAM-flexible platform for base editing and represent a versatile strategy for generating genome-edited plants with gradient-tuned agronomic traits.

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