Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA_tox, that can act upon double-stranded DNA. Here, we target DddA_tox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddA_tox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

核碱基编辑器代表了一种新兴技术，可以对真核细胞基因组进行精确的单碱基编辑。大多数核碱基编辑器使用作用于单链 DNA 的脱氨酶结构域，并需要 RNA 引导的蛋白质（例如 Cas9）在编辑前解开 DNA。然而，最新一类碱基编辑器利用脱氨酶结构域 DddA _tox ，它可以作用于双链 DNA。在这里，我们通过将这些结构域融合到工程锌指 (ZF) 阵列上，将 DddA _tox片段和基于 FokI 的切口酶靶向人类 CIITA 基因。我们还鉴定了多种与 DddA_毒素同源的毒素衍生脱氨酶 (TDD)，使我们能够微调靶向密度和特异性等特性。 TDD 衍生的 ZF 碱基编辑器可在 T 细胞中实现高达 73% 的碱基编辑，并具有良好的细胞活力和良好的特异性。