Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Nat Biotechnol. 2020 Jul;38(7):883-891. doi: 10.1038/s41587-020-0453-z. Epub 2020 Mar 16.
Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (k) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.
腺嘌呤碱基编辑器（ABE）的应用受到脱氨酶组件与 SpCas9 以外的 Cas 同源物有限兼容性的限制。我们使用噬菌体辅助非连续和连续进化（PANCE 和 PACE）对 ABE7.10 的脱氨酶组件进行了进化，得到了 ABE8e。ABE8e 包含八个额外的突变，与 ABE7.10 相比，活性（k）提高了 590 倍。ABE8e 与各种 Cas9 或 Cas12 同源物配对时，提供了大大提高的编辑效率。ABE8e 比 ABE7.10 更具连续性，这可能有利于筛选、调控区域的破坏和多重碱基编辑应用。通过在 TadA-8e 结构域中引入额外的突变，可以减轻 Cas9 依赖性和非依赖性 DNA 脱靶编辑以及转录组范围 RNA 脱靶编辑的适度增加。最后，我们表明 ABE8e 可以有效地在人类细胞中的 BCL11A 增强子或 HBG 启动子中安装上调胎儿血红蛋白表达的天然突变，而 ABE7.10 对这些靶点的编辑效果很差。ABE8e 增强了腺嘌呤碱基编辑的有效性和适用性。
