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Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors

Abstract

As the evolutionary ancestor of Cas12 nuclease, the transposon (IS200/IS605)-encoded TnpB proteins act as compact RNA-guided DNA endonucleases. To explore their evolutionary diversity and potential as genome editors, we screened TnpBs from 64 annotated IS605 members and identified 25 active in Escherichia coli, of which three are active in human cells. Further characterization of these 25 TnpBs enables prediction of the transposon-associated motif (TAM) and the right-end element RNA (reRNA) directly from genomic sequences. We established a framework for annotating TnpB systems in prokaryotic genomes and applied it to identify 14 additional candidates. Among these, ISAam1 (369 amino acids (aa)) and ISYmu1 (382 aa) TnpBs demonstrated robust editing activity across dozens of genomic loci in human cells. Both RNA-guided genome editors demonstrated similar editing efficiency as SaCas9 (1,053 aa) while being substantially smaller. The enormous diversity of TnpBs holds potential for the discovery of additional valuable genome editors.

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Fig. 1: Curation of ISfinder-annotated TnpB systems.
Fig. 2: Analyses of TAM and TnpB activity in E. coli.
Fig. 3: Identification of three active TnpB systems in human HEK293T cells.
Fig. 4: Characterization of TnpB-associated reRNA.
Fig. 5: Evolutionary and functional properties of active TnpB systems.
Fig. 6: De novo annotation and in-depth characterization of ISAam1 and ISYmu1 TnpB systems.

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Data availability

All data generated or analyzed in this study are included in this article, the supplementary information files or the dedicated databases. Small-scale datasets are directly shown in the main figures, extended data figures or supplementary tables. Sequencing data generated in this study were concurrently submitted to the NCBI Sequence Read Archive under accession number PRJNA937454 (ref. 84) and the National Genomics Data Center (part of the China National Center for Bioinformation) under accession number PRJCA015164 (ref. 85). Source data are provided with this paper.

Code availability

The codes developed for TAM depletion analysis and de novo IS605 annotation have been wrapped as two Snakemake86 workflows, accessible at both Zenodo87 and GitHub (https://github.com/Zhanglab-IOZ/TnpB).

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Acknowledgements

We thank the Barrangou laboratory for sharing the pBAD33 plasmid. We thank Wang and Zhang laboratory members for helpful discussions. We thank the HPC Platform of the Beijing Institute of Genomics for providing the computational platform. We thank B. Peng at Peking University for data processing. We thank W. Li and K. Xu for helping with AAV experiments. We thank D. Liu for helping with project management. This work was supported by the National Key Research and Development Program of China (2019YFA0110000 to H.W. and 2019YFA0802600 to Y.E.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010503 to H.W.), the Chinese Academy of Sciences (ZDBS-LY-SM005 to Y.E.Z.), the Ministry of Agriculture and Rural Affairs of China and the National Natural Science Foundation of China (31970565 to Y.E.Z. and 32101204 to G.X.). G.X. is supported by ‘ZhiYi’ Innovation Funding and the ‘ZhiYi’ Fellowship.

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H.W., Y.E.Z. and G.X. conceived, designed and supervised this work. G.X., J.S. and Y.H. performed experiments, with the help of S.C., Y. Cao., L.Y. and Y. Cai. Y.L. performed computational analysis, with the help of Y.G. Y.E.Z., H.W. and G.X. wrote the manuscript, with help from the other authors.

Corresponding authors

Correspondence to Guanghai Xiang, Yong E. Zhang or Haoyi Wang.

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A patent application has been filed by the Institute of Zoology, Chinese Academy of Sciences (H.W., G.X., J.S., Y.H., Y.E.Z. and Y.L. as inventors). The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 A schematic of the “Peel, Paste and Copy” model.

The subscripts L and R refer to the left and right ends, respectively. G refers to tetranucleotide guide sequences, while C refers to tetra- or pentanucleotide cleavage sites. CL is also the transposon insertion target site (TS) on the genome (which is marked with the unfilled bar), while GL/GR and CR are harbored by the element (marked with filled bars). The top panel shows the arrangement of IS605 locus together with the hairpin structures. During replication, TnpA catalyzes the excision (“Peel”) of transposon from the original site and the insertion (“Paste”) of transposon into a new site (the bottom left panel). Given the guidance of RNA derived from RE (reRNA, including sequences within the element as the scaffold and the downstream genome sequence as the guide), TnpB recognizes transposon-associated motifs (TAM, equivalent with CL or TS for ISDra2) and introduces a double-strand break at the original site. The break is repaired by recombining the transposon-present allele (“Copy”, the bottom right panel).

Extended Data Fig. 2 CL and TAM for 28 TnpB proteins with signals in TAM profiling experiments plus ISDra2 TnpB.

a, ISfinder-annotated CL and experimentally identified TAM sequence. CL for six conflicting cases is marked in red.

Extended Data Fig. 3 Gating strategy to determine the percentage of GFP+/RFP+ cells.

a, Cells are initially gated for viability, subsequently for singularity, and ultimately for the presence of both GFP and RFP markers (GFP+/RFP+).

Extended Data Fig. 4 Characterization of TnpB proteins.

a, Distribution of pairwise sequence identities between 65 TnpB tested. With a median identity of only 20% (marked as the red dotted line), these TnpB sequences are highly diverged. b-c,Pairwise comparison between the sequences (b) and structures (c) of archaea TnpB and those of active bacterial TnpB. Compared to the inactive TnpBs of archaea, the active TnpBs of archaea are more similar to the active TnpB systems of bacteria at both sequence and structure level. For the box plot, the figure convention follows Fig. 5c. Wilcoxon tests were used (n = 48 and 576 for the active and inactive group, respectively). d, Domain and structure of TnpB with ISTfu1 as an example. The top row shows the domain organization of ISTfu1 with 10 conserved residues marked as red lines. Herein, a fine-scale domain annotation based on knowledge of Un1Cas12f1 proteinsis shown, while a gross-scale annotation based on Pfam prediction is used in Fig. 5. The bottom panels show that the conserved N is well overlaid between ISTfu1 TnpB and Un1Cas12f1 (PDB ID: 7C7L) or Casλ (PDB ID: 8DC2). “Superposition” indicates that all three structures are overlaid. For Panels c and d, the TnpB structure is predicted with AlphaFold2.

Extended Data Fig. 5 Functional screening in HEK293T and TAM characterization of TnpB systems from de novo annotation.

a, Two TnpB systems induced reporter activity in HEK293T, as shown in Fig. 3a. Data are shown as the mean ± SD of three biological replicates with actual values overlaid. b, TAM logos for 10 TnpB systems characterized via a negative selection assay in E. coli, as shown in Fig. 2a.

Source data

Extended Data Fig. 6 Editing efficiency comparison of ISAam1, ISYmu1 and ISDra2.

Surveyor Assay gel pictures (a) and editing efficiency (b) of ISAam1, ISYmu1 and ISDra2 systems at six randomly selected endogenous sites in HEK293T cells. For Panel b, Data are shown as the mean ± SD of three biological replicates with actual values overlaid. The samples derive from the same experiment and that gels were processed in parallel.

Source data

Extended Data Fig. 7 gRNA design for seven nucleases at ten genomic loci of human.

a, Color-coded nucleases and the corresponding TAM. The gRNAs are aligned according to the stranded position. Taking CBLB as an example, the gRNA is more overlapping for ISAam1 and three Cas12f variants than for the other three nucleases. Sequences of all designed gRNAs are listed in Supplementary Table 8.

Extended Data Fig. 8 Editing efficiency of seven systems at eight genomic loci in mouse N2a cell line.

a, Distribution of the average efficiency of three biological replicates corresponding to one individual locus. The figure convention follows Fig. 6c.Ordinary one-way ANOVA test with Tukey’s multiple comparisons was performed (**P < 0.01; *P < 0.05).

Source data

Extended Data Fig. 9 Comparison of editing efficiency of ISAam1 (a) or ISYmu1 (b) relative to five Cas nucleases at three genomic loci in HEK293T cells.

The gRNA design is shown on the left panel, and editing efficiency shown on the right panel. The seven nucleases are color-coded. Since it is impossible to design overlapping gRNAs targeting the same location across all seven nucleases, two groups of overlapping gRNAs were separately designed for ISAam1, three Cas12f variants and Nme2-C.NR, and for ISAam1 and SaCas9. ISYmu1 was in a similar scenario. Data are shown as the mean ± SD of three biological replicates with actual values overlaid.

Source data

Extended Data Fig. 10 Five TnpB systems described in this study have compact size and broad editing scope in human genome.

a, Protein length distribution of representative Cas9, Cas12 and TnpB proteins. The proteins are sorted by decreasing sizes, with the dashed line marking the size of the smallest ISAam1 TnpB. Except seven proteins in Fig. 6, the widely used SpCas9 and AsCas12a together with ISDra2 TnpB and three TnpBs identified in this study are also shown. b, Protein length distribution of Cas9, Cas12 and TnpB homologs annotated in CRISPRCasdb. Notably, both Cas9 and Cas12 show a multimodal distribution, which could be caused by their intrinsic diversity in terms of protein length. In contrast, TnpB shows only one peak at approximately 400 aa. For the violin plot, the bar indicates first and third quartiles, the dot indicates the median and the curve indicates the data density. c, The proportion of potentially targetable exons in the human genome. Exons harboring at least one TAM site were counted. LincRNA refers to long intergenic noncoding RNA.

Supplementary information

Supplementary information

Supplementary Figs. 1–6 and Supplementary Protocol

Reporting Summary

Supplementary Tables 1–9

This file contains all supplementay tables.

Supplementary Data 1

This file contains source data for supplementary figures.

Source data

Source Data Extended Data Fig. 6

Unprocessed gels for Extended Data Fig. 6

Source Data Figs. 2–6 and Extended Data Figs. 5, 6, 8 and 9

Statistical source data for Figs. 2–6 and Extended Data Figs. 5, 6, 8 and 9

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Xiang, G., Li, Y., Sun, J. et al. Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors. Nat Biotechnol 42, 745–757 (2024). https://doi.org/10.1038/s41587-023-01857-x

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