工程紧型基础编辑器由AlphaFold引导的突变扫描和基于大肠杆菌的三重选择
Ryeo Gang Son1,2, Goeun Kim1, Jungjoon K Lee1,2,3
1Synthetic Biology for Clinical and Technological Innovation (SynCTI), Synthetic Biology Translational Research Programme and Department of Biochemistry, National University of Singapore, Singapore, Singapore.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
|February 3, 2026
概括
研究人员优化了一个紧的DNA除氨酶,SsdAtox,用于基编辑. 工程变体显示显著提高C-to-T转换效率和降低细胞毒性,优于现有的基础编辑器.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 基因组工程是基因组工程.
背景情况:
- 基于APOBEC1的细胞因子基编辑器是有效的,但可能因大小和细胞毒性而受到限制.
- 细菌DNA除氨酶毒素SsdAtox提供了一个较小的替代品,但效率低,毒性高.
- 基础编辑器的小型化对于高效的交付方法至关重要.
研究的目的:
- 为了改进SsdAtox,提高C-to-T基编辑效率并降低细胞毒性.
- 开发一个高通量选平台,以优化DNA脱氨酶.
- 为基因组应用程序创建紧而高效的基础编辑器.
主要方法:
- 使用AlphaFold和CASTpFold的结构导向氨酸扫描确定了SsdAtox修饰的关键残留物.
- 采用了基于大肠杆菌的新型三位一体屏幕平台的现场和突变和定向进化.
- 组合性突变发生被用来产生多位点的SsdAtox变体.
主要成果:
- 工程 SsdAtox 在 K31 守门残留物显著增加了活动,但也导致了.
- 三位一体屏幕平台成功选择了具有高活动和低度的变体.
- 优化的SsdAtox变种显示了多达31倍的改善,表现优于BE4max,并显示出较低的细胞毒性.
- 基本编辑绩效指数 (BEPI) 定义的目的是为了标准化绩效评估.
结论:
- 工程设计的SsdAtox变体代表了紧的基础编辑器技术的重大进步.
- 三位一体屏幕平台是有效的指导进化DNAdeaminases.
- 优化的SsdAtox变体为更大的基础编辑器提供了有前途的替代方案,具有更好的安全性和有效性.
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