Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity,Advanced Science

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Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity
Advanced Science ( IF 14.3 ) Pub Date : 2024-06-14 , DOI: 10.1002/advs.202309004
Shenlin Hsiao ₁ , Shuanghong Chen ₁ , Yanhong Jiang ₁ , Qiudao Wang ₁ , Yang Yang ₂ , Yongrong Lai ₂ , Tao Zhong ₁ , Jiaoyang Liao _{1,

3} , Yuxuan Wu _{1,

3}

Affiliation

The current-generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A-to-G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off-target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library-assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off-target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4–7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off-target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease-suppressing mutations and disease-correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant-library-assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.

中文翻译：

真核细胞的文库辅助进化产生具有增强编辑特异性的腺嘌呤碱基编辑器

当前一代腺嘌呤碱基编辑器（ABE）ABE8e是从原核进化系统进化而来的，在介导A到G的转化方面表现出高效，被认为在基因治疗方面具有广阔的前景。然而，其更宽的编辑窗口和更高的脱靶编辑活性限制了其在治疗用途的精确碱基编辑中的应用。这项研究采用文库辅助蛋白质进化方法，利用真核细胞生成 ABE 变体，具有更高的特异性并减少脱靶编辑，同时保持人类细胞的高活性。该研究生成了一组具有高效编辑活性的扩展 ABE，并选择了四种进化变体，与人类细胞中的 ABE8e 相比，它们在原型间隔子位置 ≈4-7 的较窄编辑窗口内提供相似或稍高的效率，这将能够最大限度地减少旁观者编辑。此外，当这些变体作为质粒或 mRNA 传递到人类细胞中时，会减少脱靶编辑事件。最后，这些变体可以有效地安装疾病抑制突变和疾病纠正突变，同时最大限度地减少不需要的旁观者编辑，这使得它们成为特定治疗编辑的有希望的方法。总之，该工作在真核细胞中建立了一种突变体文库辅助的蛋白质进化方法，并生成替代的 ABE 变体，作为精确人类基因组编辑的有效工具。

更新日期：2024-06-14

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