Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

中文翻译：

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PhieABEs：一个无 PAM/免费的高效腺嘌呤碱基编辑工具箱，在植物中具有广泛的目标范围

腺嘌呤碱基编辑器 (ABE)，通常是工程化的腺苷脱氨酶和 Cas 变体，引入位点特异性 A 到 G 突变以改善农艺性状。然而，显着不同的编辑效率、对 protospacer 相邻基序 (PAM) 的限制性要求和狭窄的编辑窗口极大地限制了它们的应用。在这里，我们通过融合进化的高活性形式的腺苷脱氨酶 TadA8e 和单链 DNA 结合结构域 (DBD)，开发了一个强大的植物高效 ABE (PhieABE) 工具箱，该结构基于无 PAM/游离链球菌化脓性识别 PAM NGN（对于 SpCas9n-NG 和 SpGn）或 NNN（对于 SpRYn）的 Cas9 (SpCas9) 切口酶变体。通过针对水稻中的 29 个代表性目标并评估结果，我们证明与 ABE7.10 和一般 ABE8e 系统相比，PhieABE 显着提高了碱基编辑活性、扩大了目标范围和更宽的编辑窗口。在这些 PhieABE 中，超 ABE8e-DBD-SpRYn (hyABE8e-SpRY) 在一些测试位点显示出接近 100% 的编辑效率，在编辑窗口中纯合碱基替换比例很高，并且没有单向导 RNA (sgRNA) 依赖的脱-目标变化。与进化的 sgRNA 相比，原始 sgRNA 与 PhieABE 更兼容。总之，DBD 融合有效地提高了碱基编辑效率，