Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector.

胞嘧啶碱基编辑能够在 DNA 中安装特定的点突变而不会出现双链断裂，并且有利于基因治疗等各种应用，但需要进一步降低脱靶风险和开发有效的递送方法。在这里，我们展示了胞嘧啶碱基编辑系统 Target-AID 的基于结构的合理工程，以最大限度地减少其脱靶效应和分子大小。通过密集和仔细的截断，其脱氨酶 PmCDA1 的 DNA 结合域被消除，并引入了额外的突变以恢复酶功能。由此产生的 tCDA1EQ 在与 Cas9 的 N 端融合 (AID-2S) 或镶嵌结构 (AID-3S) 中是有效的，显示最小化的 RNA 介导的编辑和 gRNA 依赖/独立的 DNA 脱靶，正如在人类细胞中评估的那样。