基于葡萄糖酶的基编辑器,用于哺乳动物细胞中高效的T-to-G和C-to-G编辑

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Lijun Ye^1,2,3, Dongdong Zhao^1,2,3, Ju Li⁴

¹Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.

Nature biotechnology

|January 3, 2024

概括

无去氨酶基基编辑器 (DAF-BEs) 是为了在人类遗传疾病中精确的基因编辑而开发的. 与现有方法相比,这些新的编辑器提供了更高的效率和更少的非目标效应.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
生物化学生物化学

背景情况:

当前的基准编辑器通常依赖于除氨酶,导致不受欢迎的目标外突变和编辑能力的限制.
开发更安全,更通用的基因编辑工具对于遗传疾病的治疗应用至关重要.

研究的目的:

为氨酸和氨酸基设计新的无去氨酸酶基编辑器 (DAF-BEs).
提高基准编辑的效率和特异性,以便在治疗人类遗传疾病方面有潜在的用途.

主要方法:

构建无去氨酶基编辑器 (DAF-CBE和DAF-TBE),使用氨酸-DNA或氨酸-DNA糖酶变体与Cas9尼克酶融合.
采用大肠杆菌的定向进化来产生具有改进的基转换活性的变体.
在哺乳动物细胞中测试编辑效率和非目标效应.

主要成果:

开发了DAF-CBE和DAF-TBE,提高了C-to-A和T-to-A转换效率 (分别高达58.7%和54.3%).
在哺乳动物细胞中证明了C-to-G和T-to-G编辑,显著减少了Cas9依赖和独立的目标外影响,以及最小的RNA目标外影响.
设计了DAF-CBE2/DAF-TBE2变体,具有更改的编辑窗口和改进的C-to-G/T-to-G编辑效率.

结论:

与主要编辑或CGBEs相比,DAF-BEs代表了一种新的基础编辑器类别,具有扩展的转换类型,可比的效率,更小的尺寸和减少的非目标效应.
这些无去氨酶系统为人类遗传疾病的背景下精确的基因编辑提供了有希望的替代方案.
进一步开发DAF-BEs可以扩大治疗基因编辑策略的范围.

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