Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

胞嘧啶碱基编辑器 (CBE) 是引入 C 到 T 碱基转换的有效工具，但其临床应用受到脱靶和旁观者效应的限制。通过对人类 APOBEC3A (A3A) 脱氨酶进行结构引导工程，我们开发了高度准确的 A3A-CBE (haA3A-CBE) 变体，该变体可通过狭窄的编辑窗口和接近背景水平的 DNA 和 RNA 有效地生成 C 到 T 的转换。 -目标活性，与甲基化状态和序列背景无关。工程化脱氨酶结构域与 PAM 松弛的 SpCas9-NG 变体兼容，通过单向导 RNA 的灵活定位，能够准确纠正同聚胞嘧啶位点的致病性突变。双腺相关病毒将一种 haA3A-CBE 变体递送至酪氨酸血症小鼠模型，在肝组织中诱导高达 58.1% 的编辑，且旁观者编辑最少，通过单剂量基于脂质纳米颗粒的 haA3A-信使 RNA 递送进一步减少了这种编辑。 CBE。这些结果凸显了 haA3A-CBE 在精确基因组编辑治疗人类疾病方面的巨大前景。