Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

腺嘌呤碱基编辑器 (ABE) 是新型基因组编辑工具，其活性因八个额外突变而大大增强，因此被命名为 ABE8e。然而，催化活性的提高伴随着旁观者突变的频繁产生。这种旁观者效应阻碍了其在人类基因治疗中的安全应用。为了开发催化效率高且位置精确的下一代 ABE，我们对 NG-ABE8e 进行了组合工程。我们发现了一种新的变体（NG-ABE9e），它包含九个突变。NG-ABE9e 在人类细胞中表现出强大而精确的碱基编辑活性，与 NG-ABE8e 相比，某些位点的旁观者编辑减少了 7 倍以上。为了证明其实用性，我们使用 NG-ABE9e 来纠正常染色体显性色素性视网膜炎的视紫红质中常见的 T17M 突变。它可以将旁观者编辑减少约 4 倍，同时保持相当的效率。在水稻中，NG-ABE9e 的活性显着高于 NG-ABEmax，且旁观者编辑显着低于 NG-ABE8e。因此，本研究为基因组编辑提供了一种多功能且改进的腺嘌呤碱基编辑器。