School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China.
Nat Commun. 2021 Oct 8;12(1):5897. doi: 10.1038/s41467-021-26211-0.
Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.
腺嘌呤碱基编辑器（ABE）是一种基因组编辑工具，已被用于引入精确的 A•T 到 G•C 转换。然而，ABE 在某些位点的低活性仍然是一个主要的瓶颈，限制了其有效的应用。在这里，为了解决这个问题，我们在人类细胞中开发了一种定向筛选系统来进化 ABE 的脱氨酶组件，并鉴定出三种高活性的 NG-ABEmax 变体：NG-ABEmax-SGK（R101S/D139G/E140K）、NG-ABEmax-R（Q154R）和 NG-ABEmax-K（N127K）。通过进一步的工程设计，我们创建了一个整合的变体 [NG-ABEmax-KR（N127K/Q154R）]，与原始的 NG-ABEmax 相比，它在人类细胞和小鼠疾病模型中都表现出更高的编辑活性。我们还发现，NG-ABEmax-KR 可以有效地在γ珠蛋白基因启动子中引入自然突变，编辑活性提高了四倍以上。这项工作提供了一个广泛适用、快速部署的平台，可定向筛选和进化碱基编辑器中超出增强编辑活性的用户指定特征。
