Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1. Previously, we reported a set of engineered guide RNAs supporting high indel efficiency for Cas12f1 in human cells. Here we suggest a new technology whereby the engineered guide RNAs also manifest high-efficiency programmable endonuclease activity for TnpB. We have termed this technology TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors (ABEs). A Tad–Tad mutant (V106W, D108Q) dimer fused to the C terminus of dTnpB (D354A) showed the highest levels of A-to-G conversion. The limited targetable sites for TaRGET-ABE were expanded with engineered variants of TnpB or optimized deaminases. Delivery of TaRGET-ABE also ensured potent A-to-G conversion rates in mammalian genomes. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by adeno-associated viruses, thereby harnessing the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene therapy.

转座子相关转座酶 B (TnpB) 被认为是 V 型 Cas12 家族成员的祖先蛋白，也是 UnCas12f1 的最近祖先。此前，我们报道了一组工程指导 RNA，支持人类细胞中 Cas12f1 的高插入缺失效率。在这里，我们提出了一种新技术，通过该技术，工程化指导 RNA 还表现出针对 TnpB 的高效可编程核酸内切酶活性。我们将这项技术称为TaRGET（ T npB-基于RNA的增强基因组编辑技术）。考虑到这一特点，我们建立了基于 TnpB 的腺嘌呤碱基编辑器 (ABE)。与 dTnpB (D354A) C 末端融合的 Tad-Tad 突变体 (V106W、D108Q) 二聚体显示出最高水平的 A 至 G 转化。 TaRGET-ABE 的有限靶向位点通过 TnpB 的工程变体或优化的脱氨酶进行了扩展。 TaRGET-ABE 的交付还确保了哺乳动物基因组中有效的 A 到 G 的转化率。总的来说，TaRGET-ABE 将有助于改进由腺相关病毒提供的精确基因组编辑工具，从而利用基于成簇规则间隔短回文重复 (CRISPR) 的基因治疗的发展。