Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope

Abstract

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

Fig. 1. Identification of the guide RNA and TAM required for the gene editing activity of SisTnpB7.

a sire_2474 gene (tnpB7) encoding an IS605-type TnpB is associated with an RNA spanning from the coding sequence region to the right end (reRNA). Reads coverage data were retrieved from the previously published transcriptome data. b Sequence alignment of the left end and right end sequences of IS605 transposons of S. islandicus REY15A. Conserved sequences are represented by dash lines. The insertion site of the transposon is indicated by the line above the sequence logo. c A schematic of different guide RNAs and a workflow of the gene editing experiment. RT refers to the repair template containing the homologous sequences flanking the lacS target gene. The reRNAs with the 24 nt lacS guide inserted after the most conserved region (UUCAC sequence) and the partially conserved sequence (UUCACU) were defined as the g(0) and g(1), respectively. pyrEF is a selection marker for the complementation of uracil auxotrophy. d Transformation efficiencies with different TnpB-based plasmids expressing different reRNAs targeting the same target sequence flanked by either the 5’ TTTAA or TTGAT sequence. NT is the non-targeting plasmid control. Results of gene targeting/editing plasmids on TTTAA and TTGAT motif are indicated by pink and orange filled circles, respectively. One-way ANOVA was used to compare the means of the NT and other groups based on the data obtained from three independent experiments. p values are displayed above gray lines. The differences between the means are considered statistically significant when the p-value is less than 0.05. “ns” indicates not significant (p > 0.05). e Gene editing efficiencies of different plasmids upon different TAMs. The left panel shows the inferred base pairing schemes between the guiding sequence and the target region (non-target strand). The inferred TAM sequences are underlined. The bar denotes the data mean of three biologically independent experiments. Results of gene editing plasmids on TTTAA and TTGAT motif are indicated by pink and orange circles, respectively. Source data are provided as a Source Data file.

Fig. 2. Identification of TAM variants required for the dsDNA cleavage activity of TnpB7.

a A workflow of the co-expression of TnpB7 and the lacS guide RNA, and the RNP purification from an E. coli host. IMAC, Immobilized metal affinity chromatography. SEC, Size exclusion chromatography. b Run-off sequencing result of the cleavage products. Cleavage positions at the non-targeted strand (NTS) and the target strand (TS) are marked by gray triangles. The sequences of the guide-target duplex region are shown in gray and the TAM sequence is underlined. c Time-resolved dsDNA cleavage by TnpB on TTTAA and TTTGA TAMs. The reaction system consists of 20 nM dsDNA substrates and 200 nM TnpB RNP complex. Reactions were incubated at 75 °C for 0, 1, 2.5, 5, 10, 20, 40 min and terminated by mixing with the SDS-DNA loading buffer. Control refers to a substrate that does not contain the lacS guide matching sequence. The experiment was repeated three times independently with similar results. d dsDNA cleavage activities of TnpB7 on different TAM variant sequences. Each reaction consists of 20 nM dsDNA substrates and 200 nM TnpB RNP and was incubated at 70 °C for 60 min. The value represents the fraction of substrates cleaved by TnpB (defined as the ratio of products to substrates (cleavage products + remaining substrates)). Results are shown as the mean ± SD of three replicates. Source data are provided as a Source Data file.

Fig. 3. TnpB7 facilitates gene editing on targets flanked by flexible TAM sequences.

a The left panel shows a diagram of target sites in different strains. Note that only the sequences of the non-target strand are shown. Mutated nucleotides at the TAM region are highlighted in orange. TnpB’s DNA cleavage activity on each TAM variant is indicated (as determined in Fig. 2d). The right panel indicates gene editing efficiencies of the lacS-editing plasmid (glacS-RT) in different strains. The bar denotes the data mean obtained from three biologically independent experiments. b Transformation efficiencies with TnpB-based plasmids with different strains. NT means the non-targeting plasmid. glacS means the lacS-targeting plasmid, and glacS-RT refers to the lacS gene-editing plasmid. The transformation efficiency was defined as the colony formation unit of electroporated cells per 1 µg plasmid DNA. One-way ANOVA and then Tukey test were used to compare the means of the NT and other groups based on the data obtained from three independent experiments. p values are displayed above gray lines. The differences between the means are considered statistically significant when the p-value is less than 0.05. “ns” indicates not significant (p > 0.05). c The gene editing activity of TnpB on other genomic targets flanked by weak TAM sequences. The left graph shows the transformation efficiencies with gene-targeting plasmids and gene-editing plasmids. NT means the non-targeting control. The right graph shows the gene editing efficiency results. Data were obtained from three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 4. Minimum requirements of the guiding sequence required for gene editing with TnpB.

a The minimal length of the lacS guiding sequence that supports gene editing activity of TnpB on the targets flanked by different TAMs. The lacS guide sequence was truncated from the 3’ terminal to the indicated lengths. The guide-target duplex regions are underlined. Data are obtained from three independent experiments and the bar denotes the data mean. b The effect of single base transversion in a 16 nt lacS guiding sequence on the gene editing efficiency of TnpB7. Mutated nucleotides in the TAM sequence or the guiding region are highlighted in orange. The experiment was performed on the target site flanked by either the TTTGA or TTTAA TAM. The bar denotes the data mean of three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 5. TnpB facilitates flexible single-nucleotide genome editing with templated repair.

a Gene editing efficiencies of TnpB-based SNE plasmids on the lacS target flanked by the TTTGA TAM. Mismatched positions between the lacS target and repair templates are indicated by nucleotide sequences in orange. Only the sequences of the NTS of the target region are shown. The bar denotes the data mean of three biologically independent experiments. b Gene editing outcomes with TnpB upon the reduced expression at the target flanked by the TTTAA TAM. ParaS-m38 is a ParaS promoter derivative with a reduced expression level. NT means non-targeting control. The column denotes the data mean. c Comparative analysis of SNE outcomes of TnpB on weak and strong TAM sites. Suc. and Ara. refer to sucrose and D-arabinose, respectively. Data were obtained from three biologically independent experiments. Source data are provided as a Source Data file.

Fig. 6. TnpB enables flexible genome editing in different bacteria.

a A workflow of gene editing assay with TnpB7 in E. coli. RT means a repair template. The genotypes of colonies were determined by the X-gal staining and the colony PCR. b Gene editing outcomes with TnpB in E. coli. RT1 to RT4 indicate the repair templates containing the mutated target sites. RT2-snm13 and RT3-snm8 refer to the repair template containing a single-nucleotide mutation at the 13^th and 8^th bp from the corresponding TAM sequence, respectively. Columns indicate the means of the editing efficiencies determined from three independent experiments. Source data are provided as a Source Data file. c Gene editing assay with TnpB7 in V. alginolyticus. One of representative images from three biological independent experiments is shown. Lane 1 to 8 of the gel image represents the colony PCR results for 8 randomly selected colonies from the streak plate. wt, wild type control.

Fig. 7. TAM-flexible gene editing with TnpB.

The figure depicts the relationship between targeted DNA cleavage activities of TnpB with different TAM variants and gene editing outcomes. The flexible TAM requirement of TnpB enables efficient gene deletion on strong and weak TAM sites, which greatly expands the targeting scope. However, in the case of strong TAM sites, TnpB efficiently cleaves targets containing single-nucleotide mismatches, leading to cell death. In contrast, the cleavage on the target sites or mismatched targets flanked by weak TAMs can be efficiently repaired, therefore it does not yield apparent cell death. This feature can be leveraged for efficient single-nucleotide editing with templated repair. Nevertheless, by tinkering with cellular activities of TnpB to a level that is not overwhelming to the host repair capacity, TnpB can also achieve single-nucleotide editing on strong TAM sites without inducing massive cell death. RT refers to repair template devoid of a target site or containing a mismatched target site. TAM sequences associated with an in vitro cleavage activity higher than 27% were defined as strong TAMs. TAM variants of limited gene deletion efficiencies (with an activity lower than 11.5%) are highlighted by light gray letters, the efficiencies of these TAMs in mediating gene editing can be enhanced by elevating the expression level of TnpB.