As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.

作为 Cas9 核酸酶的进化祖先，IscB 蛋白作为紧凑的 RNA 引导的 DNA 核酸内切酶和切口酶，使其成为碱基编辑的有力候选者。然而，狭窄的靶向范围限制了 IscB 系统的应用;因此，有必要找到更多识别不同靶标相邻基序 （TAM） 的 IscB。在这里，我们从在哺乳动物细胞中具有活性的未培养微生物中鉴定了 19 种未表征的 IscB 蛋白中的 10 种。通过蛋白质和 ωRNA 工程，我们进一步增强了 IscB 直系同源物 IscB.m16 的活性，并将其 TAM 范围从 MRNRAA 扩展到 NNNGNA，从而产生了一个名为 IscB.m16* 的变体。通过将脱氨酶结构域与 IscB.m16* 切口酶融合，我们生成了 IscB.m16* 衍生的碱基编辑器，该编辑器在哺乳动物细胞中表现出强大的碱基编辑效率，并通过单个腺相关病毒递送有效恢复了患病小鼠的杜氏肌营养不良蛋白。因此，本研究为基础研究和治疗应用建立了一套紧凑的碱基编辑工具。