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Hypercompact adenine base editors based on transposase B guided by engineered RNA


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.

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Fig. 1: Augment RNA-guided programmable nuclease using transposase B.
Fig. 2: Feasibility test and optimization of the TaRGET-ABE system.
Fig. 3: Expanding targetable sites by PAM variants of TnpB engineering.
Fig. 4: Switching a base-editing window by the engineering of Tad and TnpB.
Fig. 5: Validation of base-editing activity of the TaRGET-ABE-C3.0 system through AAV delivery in vitro.
Fig. 6: Assessment of base-editing activity in vivo via AAV delivery and off-target property of TaRGET-ABE system.

Data availability

Data that support the findings of this study are available within the Article and its Supplementary Information. Deep-sequencing data for large-scale validation and RNA-seq data were deposited at the NCBI Sequence Read Archive database ( under accession number PRJNA823884. All other data that support the findings of the present study and plasmid vectors are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Reditools is available at MAUND is available at


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This work was supported by a grant through the KRIBB Research Initiative Program (KGM5382221 to Y.L., D.J., K.-H.P, H.J.C, J.M.L, J.-H.K and Y.-S.K.), the National Research Foundation of Korea (INNOPOLIS) grant (2021-DD-RD-0178-01 to D.Y.K., Y.C., S.P. and Y.-S.K.), and the National Research Foundation of Korea grant (2022R1C1C1013085 to D.Y.K and S.K.) funded by the Ministry of Science and ICT, the ‘Alchemist Project’ funded by the Ministry of Trade, Industry and Energy (20012445 to D.Y.K. and Y.-S.K.), and Cooperative Research Program for Agriculture Science and Technology Development (PJ0165422022 to D.Y.K., Y.C., S.P. and Y.-S.K.) funded by Rural Development Administration.

Author information

Authors and Affiliations



Y.-S.K. conceived the study and designed the experiments. D.Y.K. and Y.C. performed overall experiments. D.J and Y.L. constructed the PAM library vectors and PAM variants of TnpB. H.J.C. and J.M.L performed base-editing assays in cells. S.P. and S.K. derived PAM-mutant HEK293T cells. K.-H.P performed the structural analysis for TnpB engineering. J.-H.K. and Y.-S.K. interpreted data. Y.-S.K. wrote the manuscript.

Corresponding author

Correspondence to Yong-Sam Kim.

Ethics declarations

Competing interests

D.Y.K., Y.C. and Y.-S.K. have filed patent applications on the TaRGET-ABE and PAM variants of TnpBthrough GenKOre. Y.-S.K. and D.Y.K. are co-founders of GenKOre. The remaining authors declare no other competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Sangsu Bae, Rahul Kohli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Construction of catalytically inactive TnpB (dTnpB) and confirmation of indel-null activity in HEK293T cells.

a. An agarose gel image showing cleavage pattern of plasmid vector by wild-type or several catalytically dead mutants of TnpB. Plasmid vectors were constructed to harbor protospacer and PAM sequence for sgRNA. Five microgram of plasmid vectors were incubated with 1 microgram of gRNA and 2 microgram of TnpB or dTnpB at 37 °C for 1 h. The cleaved vector samples were resolved on a 0.8% agarose gel. M denotes molecular ladders. The image corresponds to a representative experiment for three independent experiments. b. Indel efficiencies of TnpB or dead mutants of TnpB at an NLRC4 locus (5’-TTTAGAGGGAGACACAAGTTGATA-3’) in HEK293T cells. n = 3 independent experiments.

Extended Data Fig. 2 Dimerization of TnpB in the presence of single-stranded guide RNA.

a. An SDS-PAGE gel image for purified TnpB in elution fractions. The gel image corresponds to a representative experiment for six independent experiments. b. Size-exclusion chromatography profiles of TnpB proteins in the presence or absence of sgRNA. TnpB proteins were incubated with gRNA and the RNP complex was resolved on a Superdex 200 column. The molecular mass was estimated from a standard curve derived from the mobility of molecular ladder proteins.

Source data

Extended Data Fig. 3 Identification of the base-editing window of TaRGET-ABE-C2 system.

The substitution profile of TaRGET-ABE-C2 was explored for five endogenous sites in HEK293T cells to define a base editing window. The endogenous sites were selected from the validated sites showing substantial A-to-G conversion activities and also carrying multiple adenine sequences in the PAM-proximal region. The TaRGET-ABE system elicits A-to-G conversions in the range of positions 2–6, but most predominantly at the positions 3-4. The intensity of colors is not proportional to the editing efficiency, but highlights the qualitatively high efficiency positions.

Extended Data Fig. 4 Head-to-head comparison of TaRGET-based adenine base editors and Cas12f-based ABEMINI.

The efficiency of TaRGET-ABEs was compared with that of ABEMINI at five endogenous loci in HEK293T cells. Specifically, TaRGET-ABE-C2 was compared with ABEMINI to rule out the contribution of Tad engineering to the editing efficiency. The results show significant difference in A-to-G conversion activities in the base-editing window between TaRGET-ABE-C2 and ABEMINI, indicating TnpB as a preferred nuclease for hypercompact adenine base editors. The intensity of colors represents of the conversion efficiency. The values represent the means of three independent experiments.

Supplementary information

Supplementary information

Supplementary Figures 1–4 and Supplementary Tables 1–4.

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Source Data Fig. 5

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Source Data Fig. 6

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Source Data Extended Data Fig. 2

Unprocessed gel image.

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Kim, D.Y., Chung, Y., Lee, Y. et al. Hypercompact adenine base editors based on transposase B guided by engineered RNA. Nat Chem Biol 18, 1005–1013 (2022).

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