Introduction

IS200/IS605 transposon family is widely present in prokaryotes and eukaryotes, which can be transferred in the genome by a “peel and paste” manner1,2. Recently, TnpB encoded by the IS200/IS605 transposon family is reported to be the evolutionary ancestor of the Cas12 of type V CRISPR-Cas system3,4,5,6. TnpB processes the RNA transcript that originates from the right end (RE) of the transposon to generate ωRNA7. RNP complexes formed by TnpB and ωRNA can recognize a transposon-associated motif (TAM) and it leads to the cleaving of the target sequence which is complementary to the guide sequences at the 3′-end of the ωRNA8,9. Some TnpB nucleases have a conserved rearranged catalytic site in the RuvC-II motif, which doesn’t influence on-target cleavage, but it may regulate the collateral nuclease activity10. Some TnpBs have collateral cleavage activity9,10,11, and the collateral nuclease activity of TnpB still remains poorly understand.

Based on the RNA-guide endonuclease activity, TnpB from many species have been harnessed for genome editing and as well as in the treatment of a tyrosinaemia mouse model through AAV delivery8,12,13,14. As TnpB exhibits close evolutionary relationship with Cas12, it holds great potentiality to be applied in molecular diagnostics4,6. The collateral cleavage activity of CRISPR-Cas effectors such as Cas12a and Cas13a are well-studied and reported, but not the collateral cleavage activity of TnpB. Several sensitive, accurate, and rapid diagnostics tools have been developed based on the collateral cleavage activity of Cas12 or Cas13, such as “SHERLOCK”, “HOLMES”, “DETECTR” etc15,16,17,18. And these CRISPR-based diagnostics have been effectively improved and upgraded over the years to achieve fast, low-cost, point-of-care screening of viruses19,20,21. Limited knowledge about the collateral cleavage activity of TnpB has prevented its application in the development of TnpB-based nucleic acid detection tools.

Previously, we have reported that TnpB from Sulfolobus islandicus (SisTnpB1) could recognize the 5′-TTTAA transposon-associated motif (TAM) and cleave the DNA target specifically in vitro. Based on SisTnpB1, we have also developed a genome editing tool22. In this present study, we assessed SisTnpB1 for its collateral cleavage activity, and the results revealed that SisTnpB1 could effectively degrade single-stranded DNA (also known as trans-cleavage) under high temperature in the presence of target DNA. Mutations introduced into the TAM or seed sequences can harshly impair the trans-cleavage activity. Followingly, we applied the trans-cleavage activity of SisTnpB1 in nucleic acid detection, and it could achieve accurate detection in a rapid manner as short as 30 min. Collectively, the study revealed that the TnpB from a thermophilic archaeon could exhibit enhanced collateral cleavage activity under high temperature, and the developed molecular diagnostic tool based on it is highly sensitive.

Results

TnpB from S. islandicus exhibited high temperature activated robust trans-cleavage activity

Previously, we have reported that SisTnpB1, which is encoded by one of the tnpB genes (NCBI Gene ID: 12417531), could recognize the transposon-associated motif (TAM) of 5′-TTTAA-3′ and had the capacity to cleave both single-stranded and double-stranded DNA targets. Based on the earlier reported run-off sequencing result, we could see that the cleavage sites on the target strand is located at 20–28 nt from the TAM22. Theoretically, this cleavage pattern should produce cleavage products ranging from 28 nt to 20 nt with fluorescence, when using a 59 nt or 59 bp linear DNA with the target strand being 5′-FAM labeled (Fig. 1A and S1A). Surprisingly, unanticipated cleavage products ( > 28 nt or <20 nt) were produced by SisTnpB1-ωRNA when incubated either with single-stranded or double-stranded DNA targets (Fig. 1A and S1A). Furthermore, ssDNA without TAM but with the same guide sequence was cleaved by SisTnpB1, but that dsDNA without TAM but with the same guide sequence was scarcely cleaved by SisTnpB1 (Fig. S1B). So, we hypothesized that these ambiguous cleavage products would have come from target-activated, non-specific DNA cleavage (trans-cleavage) mediated by SisTnpB1. Then, the trans-cleavage activity of SisTnpB1 was assessed by using a single-stranded DNA (ssDNA) as the collateral substrate. The results showed that SisTnpB1 could trans-cleave ssDNA in the presence of single-stranded DNA targets, but it had higher trans-cleavage activity in the presence of the double-stranded DNA targets (Fig. 1B). So, SisTnpB1 could exhibit cis-cleavage activity to cleave the target, as well as trans-cleavage activity to degrade collateral single-stranded DNA.

Fig. 1: Determination of the trans-cleavage activity of the TnpB protein from S. islandicus REY15A.
figure 1

A Schematic for SisTnpB1-ωRNA mediated cis-cleavage of the single-stranded DNA target with TAM and denaturing PAGE analysis of the cleavage products over time (5, 10, 20, 30 min) at 75 °C. B Schematic for SisTnpB1-ωRNA mediated tran-cleavage of FAM-labelled collateral single-stranded DNA activated by single-stranded DNA targets with TAM (ssDNA target) or double-stranded DNA targets with TAM (dsDNA target) and denaturing PAGE analysis of the cleavage products over time (5, 10, 15, 20, 30 min) at 75 °C. C Denaturing PAGE analysis of SisTnpB1-ωRNA trans-cleavage activity at different temperatures. D Denaturing PAGE analysis of the trans-cleavage products generated by SisTnpB1-ωRNA at the temperatures from 66 °C to 82 °C in 30 min. Black triangles indicate the specific cleavage products (CP), and red triangles indicate the collateral cleavage products (CCP). ssDNA: collateral single-stranded DNA.

Full size image

Subsequently, we examined the trans-cleavage capability of SisTnpB1 under different temperatures. SisTnpB1 displayed robust trans-cleavage activity at 75 °C, but it exhibited highly reduced activity below 65 °C (Fig. 1C). But the trans-cleavage activity tends to increase from 66 °C to 75 °C and then tend to decline from 76 °C to 82 °C (Fig. 1D). We were curious about whether this distinct trans-cleavage activity at varying temperatures was attributed to the target binding or the target cleavage by SisTnpB1. So, the binding affinity of SisTnpB1 with ssDNA target or dsDNA target was assessed at 37 °C and 75 °C, respectively. The binding activity of SisTnpB1 with the ssDNA target was found to be higher than that with the dsDNA target at 37 °C (Fig. S1C), but no binding activity was observed at 75 °C (Fig. S1C), which contrasted with the high trans-cleavage activity at 75 °C (Fig. 1C, D). Based on this, we speculated that trans-cleavage may be related to the cis-cleavage of the target. So, we examined the cis- and trans-cleavage capabilities of SisTnpB1 with mutations in the RuvC domain. When the D187 or E271 active site in one RuvC-like domain was mutated, cis- and trans-cleavage activity were severely hindered (Fig. S1D). Overall, these results revealed that trans-cleavage activity is in company with cis-cleavage activity of SisTnpB1, and RuvC-like motifs were found to be responsible for the cleavage of not only the target, but also the collateral ssDNA.

Divalent cations and salt concentration affect the trans-cleavage activity of SisTnpB1

As we reported before, divalent cation (Mn2+ or Mg2+) was required for cis-cleavage of target for SisTnpB122. Here, we performed the trans-cleavage assay with Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Ni2+, respectively. As expected, Mn2+ or Mg2+ was found to be required for trans-cleavage activity of SisTnpB1, but it exhibited low trans-cleavage activity in the presence of Co2+ (Fig. S2A). The optimal concentration at which the trans-cleavage activity can reliably be detected is 5 mM for Mg2+, or 1 mM for Mn2+ (Fig. S2B). Additionally, trans-cleavage of the substrate was accelerated and completed within two minutes in Mn2+ buffer, compared to nine minutes in Mg2 + buffer, indicating that Mn2+ is the most effective metal ion (Fig. S2C). Although Mn2+ was proved to be the most effective metal ion for the trans-cleavage activity of SisTnpB1, it could trigger SisTnpB1 to slightly cleave ssDNA without target (Fig. S2D). Moreover, a strong inhibition in the trans-cleavage activity was observed when NaCl concentration was above 75 mM (Fig. S2E). Therefore, a buffer supplemented with 5 mM Mg2+ and 50 mM NaCl was used in all subsequent trans-cleavage and nucleic acid detection experiments. Taken together, these results indicated that the trans-cleavage activity of SisTnpB1 is Mg2+/Mn2+ dependent and is negatively affected by NaCl concentration.

Trans-cleavage activity of SisTnpB1 exhibited high sensitivity to TAM and target variations

To investigate the effects of TAM and target variations on SisTnpB1 trans-cleavage activity, double-stranded DNA targets with mismatches in the TAM or target regions were used in the trans-cleavage assay. Transversion mutations (A ↔ T or G ↔ C) were introduced into the target sequence or TAM sequence, respectively (Fig. 2A and S3A). Trans-cleavage activity was inhibited by single nucleotide mutation in TAM, but the mutation at the -5-position showed slight inhibition (Fig. 2B, C). Furthermore, the effects of single nucleotide mutation in the target on the trans-cleavage activity of SisTnpB1 was assessed by a fluorophore quencher (FQ)-labeled reporter assay (Fig. 2D). The single mutation introduced into +1 to +12 sites of the target could dramatically reduce the trans-cleavage activity of SisTnpB1 (Fig. 2E, F). Moreover, dinucleotide mutations in the first 12 base pairs of the target sequence also stopped the trans-cleavage activity (Fig. S3B and S3C), which is in line with the results of single nucleotide mutation. And the trans-cleavage activity was only slightly affected when mutations were introduced into the last 6 base pairs of the target (Fig. 2F, Fig. S3B and S3C). Altogether, our results suggested that TAM and the first twelve nucleotides of the target played a crucial role in determining the trans-cleavage activity of SisTnpB1.

Fig. 2: SisTnpB1 trans-cleavage activity is sensitive to mutations in TAM and seed sequences.
figure 2

A Diagram of single nucleotide mismatches in the TAM or target region. TAM sequences are shown in wheat and mutated sequences are highlighted in red and bold. PM: perfect match dsDNA target. B Denaturing PAGE analysis of SisTnpB1 trans-cleavage activity activated by dsDNA targets with single nucleotide mismatch in the TAM region. Black triangles indicate the collateral cleavage products (CCP). ssDNA: collateral single-stranded DNA. NT: no target control group. L: DNA ladder. C The heatmap represents the percentage of collateral cleaved products in B. D Diagram of the fluorophore quencher-labeled reporter assay activated by dsDNA targets. The ssDNA reporter is 5′-end FAM-labelled and 3′-end BHQ1-labelled (FQ), which can be cleaved by activated TnpB RNP to generate fluorescence signals. The target alone does not generate fluorescence automatically because fluorescence can only be observed when the FQ-reporter is cleaved to separate the fluorophore from the quencher. E, F Detection of the fluorescence generated by SisTnpB1 trans-cleavage of FQ-reporter. Double-stranded DNA targets with single nucleotide mutation (M1-M20) as shown in A were used to activate the trans-cleavage. PM and NT were used as the positive and negative control group. Each reaction contains 1 μM SisTnpB-ωRNA complex, 1 μM FQ-reporter, 0.5 μM dsDNA targets or equal amount of RNase-free water for the no target (NT) group. All trans-cleavage reactions were performed at 75 °C for 30 min. The fluorescence values were monitored by using a qPCR instrument. The dots represent the mean of three independent reaction replicates (n = 3) ± SEM. a.u.: arbitrary units.

Full size image

Optimal length and nucleotides of the collateral ssDNA for SisTnpB1 trans-cleavage

Next, we investigated if the collateral ssDNA affects the trans-cleavage activity of SisTnpB1 by using different lengths of ssDNA as collateral substrates. At first, we tried to find an appropriate length for SisTnpB1 trans-cleavage regardless of the sequence. So, randomly designed 44/40/32-nucleotides (nt) ssDNA with FAM-labelled were used for trans-cleavage. And the 24-nt and 18-nt ssDNA were designed by deleting some part of the 40-nt ssDNA. The results showed the 40-nt ssDNA could be completely degraded by SisTnpB1 trans-cleavage, while SisTnpB1 can barely trans-cleave the 18-nt ssDNA (Fig. 3A). Considering that the 18-nt ssDNA was part of the 40-nt ssDNA, we speculated that trans-cleavage activity of SisTnpB1 was closely related to the length of the collateral ssDNA other than the sequences. To further confirm that the 40-nt ssDNA is appropriate for SisTnpB1 trans-cleavage, we used the 40-nt and 24-nt ssDNA reporter in FQ-labeled reporter assay. SisTnpB1 could trans-cleave the 40-nt reporter to generate higher fluorescence values than that of the 24-nt reporter (Fig. 3B), indicating that ssDNA with a length of 40 nt was better for SisTnpB1 trans-cleavage.

Fig. 3: Optimal length and nucleotides of the collateral substrates for SisTnpB1 trans-cleavage.
figure 3

A Denaturing PAGE analysis of the trans-cleavage activity of SisTnpB1 with different lengths of collateral substrates. The trans-cleavage reactions were performed at 75 °C for 30 min. CCP: collateral cleavage products. B Comparison of the trans-cleavage activity of SisTnpB1 on the 24 nt and 40 nt FQ-reporters. The fluorescence values were detected by a qPCR instrument. The dots represent the mean of three independent reaction replicates (n = 3) ± SEM. a.u.: arbitrary units. C, D Comparison of the trans-cleavage activity of SisTnpB1 on FQ-reporters with different nucleotides at 75 °C for 30 min. Relative fluorescence values (ΔFluorescence) were calculated by subtracting the initial values from the final values in C, respectively. Each reaction was prepared in a total volume of 20 μL in the cleavage buffer containing 1 μM SisTnpB-ωRNA complex, 1 μM FQ-reporter, 0.5 μM dsDNA targets (or equal amount of RNase-free water for the group without target). Fluorescence can only be observed when the fluorophore is separated from the quencher by SisTnpB1 cleavage of the FQ-reporters. The fluorescence values were detected by a qPCR instrument. The dots represent the mean of three independent reaction replicates (n = 3) ± SEM. a.u.: arbitrary units.

Full size image

To explore the nucleotide preference of SisTnpB1 trans-cleavage, six collateral ssDNA with the length of 40 nt, consisting of A and T, or C and T, or G and T, or A and C, or A and G, or C and G, were used in FQ-labeled reporter assay. Remarkably, SisTnpB1 could effectively degrade collateral ssDNA which is composed of C and T or A and T (Fig. 3C). By subtracting the initial fluorescence values, cytosine and thymine (CT) collateral ssDNA in SisTnpB1 trans-cleaving generated highest relative fluorescence values (Fig. 3D). On the contrary, lowest relative fluorescence values were produced by SisTnpB1 trans-cleaving the collateral ssDNA of CG, which may be attributed to the impediment of possible secondary structure to trans-cleavage. Finally, a 40 nt FQ-labeled ssDNA consists of cytosine and thymine was chosen as the optimal collateral substrate in the following nucleic acid detection assay.

Employing the trans-cleavage activity of SisTnpB1 in nucleic acid detection

Based on the above findings, we explored whether SisTnpB1 could be applied in nucleic acid detection by using the FQ-labeled reporter assay. One plasmid with the target and the TAM sequence (pTarget), another plasmid with the target sequence and a mutated TAM (pMutant), and an empty plasmid without the target sequence (pUC19) were constructed for the nucleic acid detection assay (Fig. 4A). And serial dilutions of these three plasmids were used as the sample to be test. At first, we evaluated the detection limit of SisTnpB1 RNP for detecting the target without amplification. A significant difference in relative fluorescence values between “pTarget” group and “pMutant” group was observed when the amount of input plasmid was 20,000 pg and 2,000 pg (Fig. 4B and S4A–C). But it was not significant when the amount of input plasmid was 200 pg and 2 pg (Fig. 4B and S4A–C). Although the P value of the 20 pg group was lower than 0.05, the relative fluorescence values were too low to make the result reliable. Then, we tried to improve the sensitivity of SisTnpB1-based nucleic acid detection by combining with preamplification. Using the PCR amplified DNA as the test sample, we found that the sensitive of SisTnpB1-based nucleic acid detection method could achieve a sensitivity of 2 fg (29 cp μl−1) in 30 min (Fig. 4C and S4D–F). Based on the above results, we preliminarily determined that the trans-cleavage activity of SisTnpB1 could be employed in nucleic acid detection.

Fig. 4: Employing SisTnpB1 in fast and sensitive nucleic acid detection.
figure 4

A Flow chart of SisTnpB1-based nucleic acid detection platform. Three plasmids were used as the sample to test the detection ability of the SisTnpB1-based platform. The pTarget plasmid contains the TAM and a target sequence which can form complementary base pair with the guide sequence of ωRNA. Dinucleotides mutations were introduced into the last two nucleotides of the TAM to yield the pMutant plasmid. The empty plasmid was used as the control group (pNotarget). Equal amounts of these plasmids without preamplification or with amplification by PCR were used for detection. The detection ability was also compared by using RNP complex with SisTnpB1 incubating with in vitro transcribed ωRNA. B, C Assessment of the detection ability and sensitivity of the SisTnpB1-based platform without preamplification (B) or combined with preamplification (C). Relative fluorescence values (ΔFluorescence) were calculated by subtracting the initial values from the values at 30 min, respectively. Scatter plot represent three independent reaction replicates (n = 3) with mean ± SEM. Data were analyzed by two-tailed t-test (P < 0.05). FQ: the background fluorescence group. D Examination of the detection ability of SisTnpB1 incubating with in vitro transcribed ωRNA compared with RNP complex. “20 and 200 fg” refer to the amount of target DNA. Each reaction in B–D was prepared in a total volume of 20 μL in the cleavage buffer containing 1 μM FQ-reporter, 1 μM SisTnpB-ωRNA RNP complex or 1 μM SisTnpB1 and 1 μM in vitro transcribed ωRNA, and 1 μL of amplified target DNA or equal amount of RNase-free water. Then, the reactions were performed in a qPCR instrument at 75 °C for 60 min, followed by fluorescence detection every minute. The dots represent the mean of three independent reaction replicates (n = 3) ± SEM. a.u.: arbitrary units.

Full size image

Next, we tested if SisTnpB1 incubated with in vitro transcribed ωRNA could be used in nucleic acid detection. The detection ability of SisTnpB1 incubated with in vitro transcribed ωRNA was monitored by using 200 fg and 20 fg pTarget plasmid as the test sample. Surprisingly, we found that the fluorescence values increased slowly and reached a lower value than that of SisTnpB1 RNP at 60 min (Fig. 4D). This observation showed that SisTnpB1 incubated with in vitro transcribed ωRNA exhibited similar detection ability as RNP complex, but RNP complex takes less reaction time as short as 25 min (Fig. 4D). In addition, we explored the optimal molar ratio of ωRNA and SisTnpB1 for nucleic acid detection. The result showed that the fluorescence values increased faster and reached a comparable value to that of SisTnpB1 RNP when the molar ratio of SisTnpB1 and ωRNA was 2:1 (Fig. S5). However, the cleavage activity was hindered by incubating SisTnpB1 with either equal amount of ωRNA (1:1) or excess ωRNA (1:2) (Fig. S5). These results revealed that SisTnpB1 with in vitro transcribed ωRNA could be used in nucleic acid detection, and suggested that the trans-cleavage activity of SisTnpB1 might be repressed when excess ωRNA exists. The inhibitory effect would be reduced if TnpB and ωRNA were incubated for a sufficient time to form RNP, as the fluorescence tends to increase over time.

Taken together, we demonstrated that the SisTnpB1-based nucleic acid detection method has great potential to be used in molecular diagnostics, especially those at high temperature, and we name it “TESD” (TnpB Enable fast and Sensitive Detection). In the future, the TESD platform can be further improved by combining with isothermal amplification or other signal amplification method for one-pot testing.

Discussion

The collateral nuclease activity of TnpB from different species tends to remain ambiguous due to the evolutionary diversity of TnpB proteins10. In this study, we found that SisTnpB1 has collateral nuclease activity triggered by high temperature. There are two possible reason to explaining SisTnpB1-ωRNA’s preference for high temperatures: (1) SisTnpB1 is from a thermophilic archaea lived in high temperature, giving it natural high activity at high temperature; and (2) SisTnpB1 may have different confirmations at different temperature since high temperature can influence the structure of SisTnpB1-ωRNA. Future work could try to determine the structure of SisTnpB1 and explain the mechanistic differences for cis- and trans-cleavage. Unexpectedly, target binding was absent when trans-cleavage activity was enhanced at 75 °C. One possible reason is that the binding ability of SisTnpB1 to target is not the only factor determining its trans-cleavage activity. SisTnpB1 may be in a rapid transition state between binding and dissociation instead of stably binding to target at 75°C, and cis-/trans-cleavage may occur at the moment of binding. Furthermore, cis- and trans-cleavage activity were lost when the RuvC domain of SisTnpB1 was mutated, indicating that RuvC domain is responsible for both cis- and trans- cleavage. And we found that the collateral nuclease activity of SisTnpB1 was activated by either ssDNA targets or dsDNA targets, and was affected by the concentration of divalent cation and NaCl. These factors are equally important for the cis-cleavage activity of TnpB and Cas12f22,23. We proposed a modulation model of the cis- and trans-cleavage activity of the TnpB from thermophiles (Fig. 5). In brief, this thermal TnpB has both cis- and trans-cleavage activity. Under normal temperature, cis-cleavage activity is predominant and much higher than trans-cleavage activity. Under high temperature, the trans-cleavage activity enhanced to a level similar to cis-cleavage activity. Its high activity at high temperature suggests that SisTnpB1 can be potentially applied in nucleic acid detection.

Fig. 5: A model of the modulation for the cis- and trans-cleavage activity of SisTnpB1.
figure 5

The TnpB RNP complex has both cis- and trans-cleavage activity. Under normal temperature, the cis-cleavage activity is dominant while the trans-cleavage activity is reduced. TnpB RNP cleaves the target to generate specific length of products. Under high temperature, the trans-cleavage activity is enhanced and TnpB RNP not only cleaves the target but also degrades ssDNA without the target.

Full size image

CRISPR-based molecular diagnostics have been developed based on the collateral cleavage activity of Cas12 proteins15,24. However, only one thermostable Cas12b proteins from Brevibacillus has been characterized with activity at 70 °C, and been applied for detection25,26. As the evolutionary ancestor of Cas126, TnpB has not been applied in molecular diagnostics, especially TnpB from thermophiles. By employing optimized collateral substrates in fluorescence-quenched (FQ)-labeled reporter assays, we have preliminarily validated the ability of a TnpB from thermophilic archaea for nucleic acid detection. Moreover, the thermophilic TnpB possess robust collateral nuclease activity from 70 °C to 78 °C, which has the advantage of combining with isothermal amplification in one-pot testing. Most importantly, undetectable collateral nuclease activity at 37 °C to 65 °C of the TnpB can avoid the cleavage of the molecular beacon at the amplification stage. Furthermore, the TnpB exhibited high sensitivity to mutations at the TAM or seed sequences (-4 to +12), suggesting its further application for the detection of variant viruses.

It would be more flexible and convenient to use TnpB incubated with in vitro transcribed ωRNA in nucleic acid detection than RNP complex. The cryo-EM structure reveals that ωRNA scaffold is composed of a triplex region, a pseudoknot, four stem-loop regions, and a guide segment located at the 3′ end11,27. It has been demonstrated that ωRNA is generated by TnpB-mediated self-processing of its own mRNA, and a region of the mRNA inhibits the DNA cleavage activity of TnpB RNP complex7. In this study, we found that the collateral DNA cleavage activity of SisTnpB1 might be inhibited by excess ωRNA, which suggests that the molar ratio of TnpB and ωRNA should be taken into consideration in molecular diagnostic when using in vitro synthesized ωRNA.

In summary, our work expands the understanding of the collateral nuclease activity of a TnpB from thermophiles and make a preliminary attempt to employ TnpB in nucleic acid detection. Future work could be done to improve the performance of TnpB-based diagnostic, which make it low-cost, equipment-free, and more convenient.

Methods

Expression and purification of SisTnpB1 and RNP complex

SisTnpB1 and RNP complex were expressed and purified as described previously with minor modification22. The pET-SisTnpB1-ωRNA (Addgene plasmid no. 228365) plasmid was used for SisTnpB1 RNP production. More specifically, E. coli Rosetta (DE3) cells were cultured in LB medium supplemented with kanamycin (30 µg/ml) and chloramphenicol (50 µg/ml) at 37 °C. After the OD600 of the cells reached 0.6–0.8, protein expression was induced by adding 0.1 mM IPTG, then cultured further at 16 °C overnight. Cells were harvested and resuspended in the lysis buffer (containing 20 mM HEPES-NaOH (pH 8.0), 300 mM NaCl, 5 mM 2-mercaptoethanol, 20 mM imidazole, 2 mM PMSF, and 5% (v/v) glycerol), and lysed by high pressure. After removing cell debris by centrifugation, the supernatant was loaded onto the Ni2+-charged HiTrap chelating HP column (Cytiva, Marlborough, MA, USA). After thirty minutes of incubation, proteins were eluted with a gradient imidazole (concentration increasing from 20 mM to 500 mM) in the buffer containing 20 mM HEPES-NaOH (pH 8.0), 300 mM NaCl, 5 mM 2-mercaptoethanol, and 5% (v/v) glycerol. The fractions containing SisTnpB1 or RNP complex were collected, concentrated up to 0.8 ml and loaded onto a Superdex 200 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA). The peak fractions were analyzed by SDS-PAGE and the fractions containing SisTnpB1 or RNP complex were pooled and concentrated, then the sample was immediately used or stored at –80 °C.

Preparation of DNA substrates

Single-stranded DNA substrates with 5′-end FAM-labelled, or those with both 5′-end FAM-labelled and 3′-end BHQ1-labelled were synthesized at Sangon Biotech (Shanghai). Doubles-stranded DNA substrates used in the assays were obtained by annealing complementary oligonucleotides. Few of the oligonucleotides were 5′-end FAM-labelled for the study. The sequences of the oligonucleotides are provided in Supplementary Table S1.

In vitro DNA cleavage assay

Cleavage reactions with synthetic oligonucleotides or oligoduplex were performed at different temperatures (37–75 °C) for 60 minutes by adding 1 μM SisTnpB1 RNP complex and 0.1 μM target DNA in the buffer containing 20 mM HEPES-NaOH (pH7.5), 1 mM DTT, 1 mM EDTA, 50 mM NaCl, 5 mM MgCl2. The reaction was stopped by adding 20 mM proteinase K, 0.5 mM EDTA, and 4% SDS solution, and then it was incubated at 37 °C for 1 h. Then, 2×loading dye was added, and cleavage products were subjected to denaturing PAGE electrophoresis. DNA fragments in the gel were visualized by a FUJIFILM scanner (FLA-5100).

Collateral cleavage assay

The collateral cleavage reactions were performed by mixing 1 μM SisTnpB1 RNP complex, 0.1 μM target DNA, and 0.2 μM FAM-labelled collateral DNA in the buffer containing 20 mM HEPES-NaOH (pH7.5), 1 mM DTT, 1 mM EDTA, 50 mM NaCl, and 5 mM MgCl2 at 75 °C. For reactions with different divalent cations, 5 mM MnCl2/CoCl2/CuCl2/CaCl2/NiCl2 was used instead of MgCl2. The reactions were stopped by adding 20 mM proteinase K, 0.5 mM EDTA, and 4% SDS solution, followed by incubation at 37 °C for 1 hour. Then, 2×loading dye was added, and cleavage products were subjected to denaturing PAGE electrophoresis. DNA fragments in the gel were visualized by a FUJIFILM scanner (FLA-5100). The cleavage products were quantified using ImageJ (v.1.48) and the cleavage ratio was calculated by evaluating the percentage of the cleaved products to the total collateral substrates. The heatmaps were generated by using GraphPad Prism 8 (v.8.0.1).

DNA binding assay

The DNA binding assays were performed in buffer containing 20 mM HEPES-NaOH (pH7.5), 1 mM DTT, 1 mM EDTA, 50 mM KCl, 5% glycerol, and 50 μg/ml BSA. Each reaction contains SisTnpB1 RNP complex at different concentrations (62.5, 125, 250, and 500 nM) and 100 nM 5′-FAM-labelled DNA substrates. The reactions were incubated at 37 °C or 75 °C for 30 min, respectively, and resolved on 8% (w/v) native PAGE gels containing 0.5×TBE. After electrophoresis at room temperature for 40 min at 200 V in a 0.5×TBE running buffer, DNA in the gel was immediately visualized by a FUJIFILM scanner (FLA-5100).

Fluorophore quencher (FQ)-labeled reporter assay

The fluorophore quencher (FQ)-labeled reporter assays were initiated by mixing 1 μM SisTnpB1 RNP complex, 1 μM fluorescent-quencher (FQ) reporter, and varying amounts of trans-activating target DNA in buffer containing 20 mM HEPES-NaOH (pH7.5), 1 mM DTT, 1 mM EDTA, 50 mM NaCl, and 5 mM MgCl2 at 75 °C. The assay was performed by incubating samples in a CFX96 Touch Real-Time System (Bio-Rad). The fluorescence signal was measured for 60 min at 75 °C with measurements taken every 1 min. Each experiment was performed in triplicates. Graphs were generated by using GraphPad Prism 8 (v.8.0.1).

In vitro ωRNA transcription

Double stranded linear DNA templates containing T7 promoter were prepared by PCR using ωRNA-F and ωRNA-R (Supplementary Table S1) as the primers and pET-SisTnpB1-ωRNA as the template. Then the DNA templates were used for in vitro transcription reactions with the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) following the manufacturers’ instructions. The DNA templates were removed by adding 5 U of DNase I (RNase-free) per 1 µg of template DNA directly to a transcription reaction mixture and incubated at 37 °C for 15 min. Synthesized RNA was purified by phenol/chloroform extraction and ethanol precipitation. The RNA was used immediately for study or stored at –80 °C.

Nucleic acid detection

DNA fragments containing TAM and target sequences were obtained by annealing a pair of primers with 25 nt overlapped (Supplementary Table S1). Then the DNA fragments were cloned into the pUC19 (Addgene plasmid no.50005) by BamHI and EcoRI restriction enzyme sites to obtain the pTarget and pMutant plasmid. The pUC19 empty plasmid was used as the control (pNotarget). The concentration of these plasmids was measured by NanoDrop8000 (Thermo Scientific). Then, the plasmids were diluted in a 10-fold gradient to obtain samples to be test. To amplified the target, the PCR was performed with 1 μL of diluted plasmid DNA, 0.4 μM forward primer, 0.4 μM reverse primer (Supplementary Table S1), 0.4 mM dNTPs, 0.25 U Phanta SE Super-Fidelity DNA Polymerase (Vazyme Biotech, catalog no. P521), 12.5 µl of 2× buffer, and some RNase-free water up to a 25 μl reaction volume.

The detection mixture was conducted in a 20 μl reaction volume containing 1 μM SisTnpB1 RNP, 0.25 μM fluorescent-quencher (FQ) reporter, 1× reaction buffer composed of 20 mM HEPES-NaOH (pH7.5), 1 mM DTT, 1 mM EDTA, 50 mM NaCl, 5 mM MgCl2, and 2 μl of samples to be detected. When using in vitro transcribed ωRNA for detection, 1 μM SisTnpB1 and indicated molar ratio of ωRNA were added into the reaction instead of SisTnpB1 RNP. The reactions were incubated at 75 °C for 30 min or 60 min, and the fluorescence values were collected by a CFX96 Touch Real-Time System (Bio-Rad). Relative fluorescence values were calculated by subtracting the initial values from the final values. Each experiment was performed in triplicates.

Statistics and Reproducibility

All the statistical data were calculated using GraphPad Prism 8 (GraphPad Prism Software Corporation, San Diego, CA). The unpaired t-test (two-tailed) was used to determine significant differences. Data from at least three biologically independent experiments are shown as mean value ± Standard Error of the Mean (SEM). The P-value  <  0.05 was judged as the statistical significance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.