Transposon-associated transposase B (TnpB) is deemed an ancestral protein for type V, Cas12 family members, and the closest ancestor to UnCas12f1 due to its high sequence similarity. Previously, we reported a set of engineered guide RNAs supporting high indel e ciency for Cas12f1 in human cells. Here, we suggest a new technology whereby the engineered gRNAs also manifest highly e ciency programmable endonuclease activity for TnpB, termed TaRGET (TnpB-augment RNA-based Genome Editing Technology). Having this feature in mind, we established TnpB-based adenine base editors. A codon-optimized 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 by either developing several PAM variants of TnpB or by engineering TnpB and optimizing deaminases at PAM-distal and PAM-proximal sites, respectively. When delivered by AAV, the TaRGET-ABE showed potent A-to-G conversion rates in human cells. Collectively, the TaRGET-ABE will contribute to improving precise genome-editing tools that can be delivered by AAV, thereby harnessing the development of CRISPR-based gene therapy. MainLiving organisms are subjected to spontaneous genetic variations, which form the basis for biodiversity and evolution. The random nature of genetic variation as well as non-biological factors are also responsible for a variety of genetic disorders. Of the types of genetic variations identi ed in humans, single-nucleotide variations (SNVs) account for nearly half of disease-related mutations 1 . This suggests that the development of site-speci c, precise genome editing tools holds promise for the treatment of otherwise intractable genetic disorders. CRISPR/Cas-mediated base-editing systems have addressed the unsatisfactory modi cation e ciency of homology-directed repair (HDR) by exploiting the high genetargeting capability of the CRISPR/Cas systems. The catalytically inactive Cas (dCas) or nickase Cas (nCas) fused to naturally occurring or engineered deaminases led to highly e cient single-nucleotide alterations including the C:G-to-T:A 2 and A:T-to-G:C 3 conversion. The introduction of an E. coli-derived uracil DNA N-glycosylase also enabled C:G-to-G:C transversion 4 . Moreover, the base editing systems guarantee higher levels of safety from a clinical view point because they enable precise genome editing with negligible or low levels of double-strand breaks (DSBs). In addition to such innate high e ciency and safety, base editing systems have further evolved in various aspects including decreased off-target editing, enhanced conversion speci city, broadened editing windows, increased editing e ciency, and control of unwanted editing [5][6][7] .Despite the dramatic improvements of base-editing system per se, the challenges on the delivery side remain a major hurdle preventing base editors from being widely used in clinical applications 8 . Adenoassociated viruses (AAVs) are considered as a validated delivery platform due to the...

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