Main

CRISPR–Cas systems in prokaryotes provide adaptive immunity against foreign nucleic acids, and are divided into two classes (classes 1 and 2) and six types1,4 (types I–VI). The class 2 systems include types II, V and VI, in which Cas9, Cas12 and Cas13, respectively, function as effector enzymes responsible for interference. Type II Cas9 effector proteins associate with dual RNA guides (CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) or their artificially connected single-guide RNA (sgRNA)) and cleave double-stranded DNA (dsDNA) targets using the HNH and RuvC nuclease domains5,6. Type V Cas12 effector proteins are further divided into Cas12a–m subtypes and span diverse functionalities7,8,9,10,11,12,13,14,15. Although the Cas12 proteins commonly have a single RuvC nuclease domain, they share low sequence similarity outside this conserved region. Cas12 enzymes associate with either crRNA guides or dual RNA guides (crRNA and tracrRNA) to cleave dsDNA targets, using their single RuvC domain. Recent reports have demonstrated that the class 2 CRISPR effectors Cas9 and Cas12 evolved independently from two members of the IS200/IS605 transposon-encoded nuclease superfamily, IscB and TnpB, respectively1,2,3. Functional and structural studies of IscB revealed that an associated ωRNA (obligate mobile element guided activity (OMEGA)) has a crucial role in recognizing the guide RNA–target DNA heteroduplex, enabling IscB to cleave its target DNA using the HNH and RuvC domains16,17.

TnpB proteins are also RNA-guided DNA endonucleases2,3. TnpB associates with a single, long non-coding RNA (referred to as ωRNA, also known as right end element RNA (reRNA)). The gene encoding the ωRNA overlaps with the 3′ end of the tnpB gene and the non-coding right end element of the transposon (Fig. 1a), and TnpB cleaves dsDNA targets complementary to the ωRNA guide sequence by using its RuvC domain. In addition, TnpB requires a target-adjacent motif (TAM) upstream of the target sequence to cleave the target DNA, similar to the Cas enzymes, which require a protospacer-adjacent motif (PAM) sequence to cleave their target DNAs18. TnpB from D. radiodurans ISDra2 (hereafter referred to as TnpB for simplicity) consists of 408 residues (Extended Data Fig. 1) and has a similar domain organization to Cas12f, which is the smallest subtype among the type V Cas12 enzymes11. However, a recent molecular mass analysis revealed that TnpB functions as a monomer, whereas Cas12f functions as a dimer3,19. Moreover, whereas Cas12f associates with dual RNA guides, TnpB associates with the single ωRNA. Therefore, how the compact TnpB protein assembles with its cognate ωRNAs to mediate the RNA-guided double-stranded DNA breaks remains unknown.

Fig. 1: Cryo-EM structure of the TnpB–ωRNA–target DNA ternary complex.
figure 1

a, Schematic of the D. radiodurans ISDra2 locus. The MGE consists of the tnpA and tnpB genes flanked by the left end (LE) and right end (RE) elements of the transposon. The ωRNA is derived from the 3′ end of the tnpB gene and the RE element. b, Diagram of the ωRNA and target DNA used for cryo-EM analysis. The target strand (TS) and non-target strand (NTS) each comprise 35 nucleotides, and the non-target strand contains a TTGAT TAM sequence. The 247-nt ωRNA was co-expressed and co-purified with TnpB. Nucleotides −231 to −117, −70 to −49, −20 to −17 and 13 to 16 of the ωRNA, nucleotides −8 to 4 and 27 of the target strand, and nucleotides −11* and 1* to 24* of the non-target strand were not included in the final model. c, The domain structure of TnpB. CTD, C-terminal domain. Residues 281 to 296 and 379 to 408 were not included in the final model. d, Cryo-EM density map of the TnpB–ωRNA–target DNA complex. e, The overall structure of the TnpB–ωRNA–target DNA complex. Disordered regions are indicated as dotted lines.

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Structure of TnpB–ωRNA–target DNA

To elucidate the molecular mechanism of TnpB, we co-expressed TnpB and its cognate 247-nucleotide (nt) ωRNA containing a 16-nt guide segment at the 3′ end, and then purified the TnpB–ωRNA complex. We reconstituted the ternary TnpB–ωRNA–target DNA complex by mixing the purified TnpB–ωRNA complex and a 35-base pair (bp) double-stranded DNA with phosphorothioate modifications within the DNA backbone around the cleavage site and the TTGAT TAM sequence, and analysed its ternary structure by cryo-EM (Fig. 1b). We obtained a three-dimensional reconstruction of the ternary complex with an overall resolution of 3.2 Å (Fig. 1c–e, Extended Data Fig. 2, Extended Data Table 1 and Supplementary Fig. 1). The cryo-EM structure revealed that a single TnpB molecule assembles with a single ωRNA molecule to form a ribonucleoprotein effector complex, consistent with the previous molecular mass analysis3. TnpB adopts a bilobed architecture consisting of recognition (REC) and nuclease (NUC) lobes, which are connected by a linker loop (Fig. 1c–e). The REC lobe comprises the wedge (WED) and REC domains, and the NUC lobe consists of the RuvC nuclease domain and the target nucleic acid-binding (TNB) domain. The C-terminal domain (residues 376 to 408), which has little sequence homology among TnpB proteins, is disordered in the present structure except for its first three residues (Extended Data Fig. 1). The ωRNA–target DNA heteroduplex is accommodated within a central channel formed by the WED, REC and RuvC domains (Fig. 1d,e and Extended Data Fig. 3a). The TAM-containing DNA duplex (the TAM duplex) is bound to the cleft formed by the WED and REC domains, whereas the ωRNA scaffold binds to a surface formed by the WED and RuvC domains (Extended Data Fig. 3b–e). In the interior of the complex, the amino acids of TnpB and the nucleotides of the ωRNA and the target DNA are clearly visible in the density map. By contrast, we did not observe clear densities for the peripheral regions, such as the C-terminal domain (residues 379 to 408), ωRNA scaffold (nucleotides −70 to −49) and the TAM-distal DNA duplex (re-hybridized duplex), indicating the flexibility of these regions. Thus, residues 281 to 296 and 379 to 408 of TnpB, nucleotides −231 to −117, −70 to −49, −20 to −17 and 13–16 of the ωRNA, nucleotides −8 to 4 and 27 of the target strand, and nucleotides −11* and 1*–24* of the non-target strand were not included in the final model.

Domain structure

The WED domain (residues 1 to 12 and 117 to 176) comprises a seven-stranded β-barrel flanked by an α-helix and adopts an oligonucleotide/oligosaccharide-binding fold (Extended Data Fig. 4a). The REC domain (residues 13 to 116) is inserted between the β1 and β2 strands of the WED domain and is composed of four α-helices. The RuvC domain (residues 185 to 326 and 360 to 375) has an RNase H fold, consisting of a five-stranded mixed β-sheet flanked by four α-helices. The RuvC domain of TnpB structurally resembles those of Cas12 enzymes, and the conserved D191, E278 and D361 residues form a catalytic centre similar to those of other Cas12 enzymes19,20,21 (Extended Data Fig. 4a). The TNB domain (residues 327 to 359) is inserted between the β5 strand and the α4 helix and contains a CCCC-type zinc-finger, in which a zinc ion is coordinated by four conserved cysteine residues (C331, C334, C351 and C354) (Extended Data Fig. 4a,b). A structural comparison of TnpB with Cas12 enzymes revealed that TnpB represents the minimal domain organization common to all Cas12 enzymes19,20,21 (Extended Data Fig. 4a).

Characterization of the ωRNA

In the present structure, we observed a density for the 3′ end of the ωRNA (−116G to 16C), except for the peripheral region, whereas we could not detect a density for the 5′ end (−231G to −117T) (Fig. 2a,b and Extended Data Fig. 3c). Consistent with this observation, a denaturing gel analysis revealed that the purified TnpB was bound to 100–160 nt of the ωRNA (Extended Data Fig. 5a), even though it was co-expressed with a 247-nt ωRNA. To understand which part of the ωRNA sequence remains bound to the TnpB protein, we performed northern blotting analyses using five DNA probes (I–V) covering the entire ωRNA sequence (Extended Data Fig. 5b,c and Supplementary Fig. 2). In cells expressing ωRNA only, the total RNA did not yield any ωRNA band, strongly suggesting that the ωRNA is degraded by endogenous RNases in the absence of the TnpB protein (Extended Data Fig. 5c). By contrast, in cells co-expressing ωRNA and TnpB, three different RNAs, approximately 220, 160 and 130 nt in size, were found in the total RNA extract (Extended Data Fig. 5c). The approximately 220-nt RNA was detected by probes II–V and the approximately 160 and 130-nt RNAs were detected by probes III–V, respectively, indicating that these RNAs commonly contain the guide sequences on the 3′ end. The approximately 160 and 130-nt RNAs were also observed in the RNA extracted from the purified TnpB–ωRNA complex, whereas the approximately 220-nt RNA was barely observed, suggesting that the 220-nt RNA was degraded during the purification process or urea gel electrophoresis (Extended Data Fig. 5c). Next, we isolated the approximately 160-nt and 130-nt RNAs and performed liquid chromatography-mass spectrometry (LC–MS) analyses to determine the processing sites of these RNAs. The approximately 160-nt RNA treated with RNase A had a pGAACp fragment (Extended Data Fig. 5d), suggesting that this RNA was cleaved between −150A and −149G or −138U and −137G by TnpB and/or endogenous RNases. We could not identify the cleavage site of the approximately 130-nt RNA, since the region around −110 to −120 is difficult to analyse owing to its GU-rich sequence. However, we did observe a clear density for −116G, but not −117T, suggesting that the approximately 130-nt RNA is cleaved between −117T and −116G. We concluded that the ωRNA is processed by TnpB or endogenous RNases at multiple sites at the 5′ end, and that at least the 130-nt fragment including the guide at the 3′ end of the ωRNA remains stably bound to the TnpB protein.

Fig. 2: ωRNA architecture.
figure 2

a, Schematic of the ωRNA and target DNA. Nucleotides −231 to −117, −70 to −49, −20 to −17, and 13 to 16 of the ωRNA, nucleotides −8 to 4 and 27 of the target strand, and nucleotides −11* and 1* to 24* of the non-target strand were disordered and not included in the model. These disordered regions are enclosed in grey boxes. b, Structure of the ωRNA scaffold. The disordered regions are indicated as dotted lines. c, Insertion–deletion mutation (indel) formation efficiencies of TnpB with wild-type (WT) ωRNA, ωRNA with deleted 5′ region (Trim1), ωRNA lacking both 5′ region and stem 3b (Trim2) and non-targeting ωRNA (NT) at seven endogenous target sites in HEK293FT cells. Data are mean ± s.d. (n = 3 biologically independent samples).

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Co-evolution of the tnpB gene and ωRNA

Previous studies revealed that many archaea and bacteria transcribe non-coding RNAs (ncRNAs) overlapping the 3′ end of the tnpB gene, suggesting that these overlapping ncRNAs have conserved roles in prokaryotes2,22. Indeed, the ωRNA of ISDra2 TnpB overlaps the 3′ end of the tnpB gene (residues 335 to 408 and −231G to −10U) (Extended Data Fig. 6a). However, our structural and biochemical analyses showed that the ωRNA bound to the TnpB was processed on the 5′ side, as described above. Indeed, the truncation of the 5′ region of the ωRNA (Δ−231G to −117T (Δ5′ region)) had no effect on the TnpB-mediated DNA cleavage, indicating that this region of the ωRNA (−231G to −117T) is not required for cleavage (Extended Data Fig. 6b,c). Conversely, the C-terminal domain (residues 376 to 408) has relatively low sequence homology among TnpB proteins (Extended Data Fig. 1), and is disordered in the present structure. Our in vitro cleavage assay revealed that a C-terminal truncation mutant (Δ376 to 408 (ΔCTD)) efficiently cleaves the target DNA, although the protein stability is slightly decreased compared with the wild-type TnpB (Extended Data Fig. 6b–d). This result indicated that the C-terminal domain is not required for the RNA-guided target DNA cleavage among TnpB proteins. Thus, our structure revealed that the TnpB C-terminal region (residues 376 to 408 overlapping with −109G to −10U) is disordered and not involved in the DNA cleavage, whereas the 5′ region of the ωRNA (−231G to −117T, overlapping with residues 336 to 373) is not crucial for the target DNA cleavage. Therefore, except for a few nucleotides, the functionally important regions of the tnpB gene and ωRNA do not overlap, suggesting that although ωRNA expression and processing may require co-expression with the TnpB protein, the co-evolution of these two elements is less constrained than previously predicted and avoids the overlap of functionally essential gene regions (Extended Data Fig. 6a).

ωRNA architecture

The present structure demonstrated that the TnpB–ωRNA scaffold adopts an unexpected architecture compared with that expected from the primary sequence2 (Fig. 2a,b). The ωRNA (−116G to 16C) consists of the 16-nt guide segment and 116-nt RNA scaffold, comprising four stems (stems 1–4) and a pseudoknot (PK). Notably, −5U to −3C base pair with −103A to −105G, rather than with the predicted −30A to −32G, and −6G and −2A form non-canonical base pairs with −102U and −106G, respectively, to construct the PK (Fig. 2a,b and Extended Data Fig. 3c). The PK coaxially stacks with stem 1 to form a continuous helix. Nucleotides −35U to −32G base pair with −81A to −84C, and −36G and −31A form non-canonical base pairs with −80U and −85G, respectively, to form stem 2 (Fig. 2a,b and Extended Data Fig. 3d). In addition, −91U to −86C base pair with −111C to −116G in stem 1, thereby contributing to the triple helix formation (Fig. 2a,b and Extended Data Fig. 3e). As expected from the nucleotide sequence, stem 3a contains a 7-bp duplex—pairing −77A to −71U with −42U to −48A—with a loop, whereas stem 3b (−70G to −49A) is unresolved, suggesting the intrinsic flexibility of this region (Fig. 2a,b and Extended Data Fig. 3d).

Previous studies demonstrated that a truncation of the disordered region of the Cas12f sgRNA improved genome editing efficiency in mammalian cells23,24. To test this idea in TnpB, we constructed an ωRNA truncated mutant, in which the 5′ end of the ωRNA (nucleotides −231G to −117T) was deleted (referred to as Trim1). The Trim1 mutant induced indels at efficiencies similar to or higher than those of the full-length ωRNA (Fig. 2c). An additionally truncated mutant, in which stem 3b (−70G to −49A) was deleted by connecting −71U and −48A with a GAAA linker (referred to as Trim2), exhibited further enhanced genome editing activity (Fig. 2c). These results confirmed the utility of TnpB in combination with the Trim2 ωRNA as a compact genome-engineering tool.

A structural comparison of the ωRNA with the guide RNAs of Cas12 enzymes revealed the presence of a structurally conserved core region19,20 (Extended Data Fig. 7a). The guide RNAs of Cas12 enzymes commonly have PK structures preceding the guide sequences, despite their distinct architectures, whereas the ωRNA of TnpB forms the conserved PK structure. These core regions are recognized by their cognate TnpB and Cas12 proteins in similar manners (Extended Data Fig. 7b) (described below). A structural comparison of the ωRNA with the sgRNA of Cas12f also revealed similarities between them. The guide, PK and stem 4 regions of the ωRNA structurally resemble the guide, PK (repeat–antirepeat 1) and stem 5 (repeat–antirepeat 2) regions of the Cas12f sgRNA (Extended Data Fig. 7a). Furthermore, other regions of the ωRNA form similar stem loops to those of the Cas12f sgRNA and interact with the TnpB protein. Therefore, our structure revealed that the ωRNA functions as a ‘natural’ sgRNA, in which a crRNA-like region and a tracrRNA-like region are connected by a UUUA tetraloop (−20U to −17A) (Extended Data Fig. 7c).

ωRNA recognition

TnpB recognizes the ωRNA via its WED and RuvC domains, mainly through interactions with its sugar-phosphate backbone (Fig. 3a and Extended Data Fig. 8a). The stem 1 and triple helix structure of the ωRNA are recognized by the α1 and α2 helices of the RuvC domain through electrostatic interactions between its sugar-phosphate backbone and highly conserved basic residues (Fig. 3b). Stems 2 and 3a extensively interact with the RuvC domain (Fig. 3c and Extended Data Fig. 8a). Nucleotides −36G, −73U and −74C form hydrogen-bonding interactions with R232, Q227 and R231 respectively, and the nucleobase of −35U forms a stacking interaction with R238 (Fig. 3c). Stem 4 is recognized by the WED domain primarily through electrostatic interactions between its upper stem region and strands β4 and β5 of the WED domain (Fig. 3d). The conserved PK region of the ωRNA is sandwiched between and extensively recognized by the WED and RuvC domains (Fig. 3e and Extended Data Fig. 7b). The −105G:−3C base pair in the PK is recognized by K263 through a hydrogen bond. The nucleobase of −1A between the PK and the guide segment is sandwiched by Q148 and the −106G:−2A non-canonical base pair, and the ribose moiety of −1A forms a hydrophobic interaction with H262 (Fig. 3e). Notably, Cas12 enzymes recognize the conserved PK structure in their cognate guide RNAs by the WED and RuvC domains in a similar manner19,20 (Extended Data Fig. 7b), consistent with the notion that the core regions of the ωRNA and the guide RNAs are highly conserved among the TnpB and Cas12 enzymes and are important for the target DNA cleavage.

Fig. 3: ωRNA recognition.
figure 3

a, Recognition of the ωRNA scaffold by the TnpB protein. TnpB is shown as a surface model. The ωRNA scaffold is recognized mainly through the WED and RuvC domains. b–e, Recognition of the stem 1 and triple helix structure (b), stems 2 and 3a (c), stem 4 (d) and the PK architecture (e) of the ωRNA scaffold. Hydrogen bonding and electrostatic interactions are shown as dashed lines.

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TAM recognition

The TTGAT TAM duplex is recognized by the WED and REC domains (Fig. 4a and Extended Data Figs. 3b and 8a). The nucleobase of −1*dT in the non-target strand forms van der Waals interactions with Y52 and S56 in the REC domain (Fig. 4b). The nucleobase of −2*dA in the non-target strand forms a hydrogen bond with Q80 in the REC domain, and the methyl group of dT18 in the target strand forms van der Waals interactions with F77 in the REC domain (Fig. 4b). O6 and N7 of −3*dG in the non-target strand form a bidentate hydrogen bond with the side chain of K76 in the REC domain, which is anchored via a stacking interaction with F77 (Fig. 4c). The 5′-methyl group of −4*dT forms van der Waals interactions with the side chains of F77 in the REC domain and T123 in the WED domain, whereas the nucleobase of −5*dT hydrogen bonds with N124 in the WED domain (Fig. 4c). The Y52A, K76A, Q80A and T123A mutations abolished the cleavage activity of TnpB, whereas the S56A, F77A and N124A mutations substantially reduced the DNA cleavage activity of TnpB, confirming the functional importance of these residues for the TAM recognition (Fig. 4d). Together, our structural and functional analyses revealed that TnpB forms sequence-specific contacts with both target and non-target strands to achieve TAM recognition.

Fig. 4: Target DNA recognition and loading.
figure 4

a, Recognition of the target DNA. TnpB is shown as an electrostatic surface potential model. The TAM duplex is bound to the cleft formed by the WED and REC domains. The guide RNA–target DNA heteroduplex is accommodated within a positively charged central channel formed by the REC and RuvC domains. b,c, Recognition of the TAM duplex. Nucleotides −1*dT, 18dT and −4*dT and residues Y52, S56, F77 and T123 are depicted by space-filling models. Hydrogen bonding and electrostatic interactions are shown as dashed lines. d, In vitro DNA cleavage activities of the wild-type TnpB and TAM recognition mutants with ωRNA with deleted 5′ region. The 3-kb linearized target DNA containing the 16-nt target sequence was incubated with the TnpB–ωRNA complex (250 nM) at 37 °C for 30 min. The reaction products were resolved, visualized and quantified with a MultiNA microchip electrophoresis device. Data are mean ± s.d. (n = 3 biologically independent samples). The experiments were repeated three times with similar results. e,f, Recognition of the TAM-proximal region of the guide RNA–target DNA heteroduplex (e) and the TAM-distal region of the heteroduplex (f). Hydrogen bonding and electrostatic interactions are shown as dashed lines. g, Positions of the active site and the target DNA. The possible trajectories of the target strand are shown by a dashed arrow. h, In vitro DNA cleavage activities of TnpB with five target DNAs with different sequences at the re-hybridized DNA duplex. Re-hybridized regions with altered sequences are highlighted with a yellow background. CG and AT sequences are coloured blue and green, respectively. Data are mean ± s.d. (n = 3 biologically independent samples). The experiments were repeated three times with similar results.

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Target DNA recognition

The guide RNA–target DNA heteroduplex is accommodated within a positively charged central channel formed by the REC and RuvC domains and is recognized through interactions with its sugar-phosphate backbone (Fig. 4a and Extended Data Figs. 3a and 8a). The backbone phosphate group between dA17 and dC16 in the target strand is recognized by Q121 and N156 in the WED domain, and the first 1G:16dC base pair stacks with N4 and Y309/W313 in the WED and RuvC domains, respectively (Fig. 4e), as also observed in Cas12 enzymes20. These interactions facilitate the target DNA unwinding and the guide RNA–target DNA heteroduplex formation. The displaced single-stranded non-target strand in the target dsDNA is barely visible in the present structure, owing to its flexibility. In the guide–target heteroduplex, (1G to 11G):(16dC to 6dC) of the TAM-proximal region are accommodated within the positively charged central channel and recognized by TnpB through electrostatic interactions with its sugar-phosphate backbone (Extended Data Fig. 8a). By contrast, the TAM-distal region in the heteroduplex (12C:3dG) is exposed to the solvent, and the four terminal base pairs (13G to 16C):(4dC to 1dG) are disordered (Fig. 4f). These structural observations suggested that the base pairs in the TAM-distal region are not recognized by TnpB. Indeed, our in vitro cleavage assays demonstrated that TAM-proximal double mismatches (positions 1–12) abolish the TnpB-mediated target DNA cleavage, whereas TAM-distal double mismatches (positions 13–16) reduce but still allow the target DNA cleavage by TnpB (Extended Data Fig. 8b). In addition, we analysed the target DNA specificity by performing a genome-wide off-target analysis for TnpB in human cells, and found many off-target sites with mismatches in the TAM-distal region25 (Extended Data Fig. 8c). These results indicated that the TAM-proximal 12 bp (approximately) of a guide RNA–target DNA heteroduplex is important for the specificity of the RNA-guided target DNA cleavage by TnpB proteins.

DNA cleavage mechanism

Previous studies revealed that TnpB cleaves the target and non-target strands at 21 nt and 15–21 nt downstream of the TAM, respectively, using a single RuvC active site3. Cas12 enzymes recognize the PAM-distal region of the guide RNA–target DNA heteroduplex and the re-hybridized DNA duplex by their TNB domains, which facilitate the DNA unwinding and loading into the RuvC active site19,20,21,26 (Extended Data Fig. 8d). By contrast, in the TnpB structure, the TAM-distal region of the heteroduplex is located far from the TNB domain, and the end of the TAM-distal region of the heteroduplex and the re-hybridized DNA duplex is disordered (Fig. 4g). These observations suggested that TnpB, unlike Cas12 enzymes, does not interact with these regions. Thus, we hypothesized that TnpB is unable to unwind the re-hybridized DNA by itself, but instead relies on spontaneously unwound DNA. To test this hypothesis, we performed an in vitro cleavage assay with five target DNAs with different sequences at the site of the DNA duplex that should be re-hybridized (Fig. 4h). We found that TnpB cleaves the target DNAs with AT-rich sequences more efficiently than those with GC-rich sequences (Fig. 4h). These results support the idea that TnpB spontaneously cleaves the unwound target DNA and does not unwind the target DNA by itself.

Discussion

In this study, we determined the cryo-EM structure of the TnpB–ωRNA–dsDNA ternary complex, revealing how the relatively small TnpB protein recognizes its cognate ωRNA to form a compact effector complex that can cleave a dsDNA target that is complementary to the ωRNA guide sequence. Our structural and functional analyses revealed that TnpB requires the formation of a heteroduplex between an approximately 12-bp guide RNA and target DNA to mediate DNA cleavage and tolerates TAM-distal mismatches, suggesting that TnpB has several target sites in its own host genome. These observations indicated that TnpB may be involved in transposon propagation as well as transposon homing, although further biochemical analyses are needed to fully characterize the function of TnpB in transposition.

A structural comparison of TnpB with Cas12 enzymes highlighted the conservation of the molecular mechanism during their evolution from a key role in guided transposition to one in adaptive immunity19,20,21 (Extended Data Fig. 9). TnpB and Cas12 enzymes share a bilobed architecture consisting of the REC and NUC lobes. By contrast, the Cas9 ancestors IscB and IsrB lack a REC domain and instead use their bulky cognate ωRNAs for an analogous role to the REC domains in Cas916,17,27 (Extended Data Fig. 10a). TnpB and Cas12 enzymes also recognize the TAM–PAM duplex in the groove formed by the WED and REC domains, facilitating the initial DNA unwinding and the guide RNA–target DNA heteroduplex formation. The structural comparison also revealed mechanistic differences for the target DNA loading, although the TnpB and Cas12 enzymes cleave the target and non-target strands at the single RuvC site. Upon cleavage of the non-target strand, Cas12 enzymes unwind the re-hybridized DNA duplex and load the target strand into the RuvC active site, whereas TnpB is unable to unwind the re-hybridized DNA duplex and captures spontaneously unwound DNA near the RuvC active site. Furthermore, the structural comparisons with two different types of compact Cas12 enzyme, UnCas12f1 and MmCas12m2 (also known as Cas12U-1), which show relatively high sequence similarity to TnpB proteins, provided insights into the evolution of type V CRISPR–Cas12 effectors15,19 (Fig. 5 and Extended Data Fig. 10b). In the present structure, TnpB uses its REC and RuvC domains to recognize a relatively short approximately 12-bp heteroduplex (Fig. 5 and Extended Data Fig. 9). Although UnCas12f1 has a similar domain organization to TnpB, it functions as a dimer to interact with an approximately 20-bp heteroduplex, with the second molecule recognizing the terminal 6 bp of the heteroduplex (Fig. 5 and Extended Data Fig. 9). By contrast, although MmCas12m2 functions as a monomer, it uses a characteristic kinked coiled-coil insertion in the REC domain (referred to as the REC2 domain), in addition to the REC and RuvC domains, to recognize an approximately 17-bp heteroduplex (Fig. 5). All Cas12 enzymes, except for Cas12f and Cas12k (which are associated with one or more additional protein molecules), have the REC2 insertion to allow the recognition of the PAM-distal region (Extended Data Fig. 9). These structural findings suggest that Cas12 enzymes acquired the ability to recognize the PAM-distal end of the guide RNA–target DNA heteroduplex through two distinct strategies, either by dimerization or by REC2 insertion, to achieve the target specificity required for CRISPR–Cas adaptive immunity via the increased length of the effective guide (Fig. 5). The development of these different mechanistic strategies from a common minimal TnpB scaffold for the same ultimate function suggested the distinct evolutionary origins for the different Cas12 lineages1. Therefore, these findings indicate that the evolutionary path of Cas12 enzymes from TnpB is distinct from that of Cas9 enzymes from IscB, as Cas12 may have arisen from TnpB on multiple independent occasions, in contrast to the single evolutionary event that probably gave rise to all extant Cas9 variants.

Fig. 5: Comparison of TnpB with Cas12f and Cas12m.
figure 5

Structural comparison of TnpB with Cas12f (Cas12f from an uncultured archaeon) (PDB ID: 7C7L) and Cas12m (Cas12m from Mycobacterium mucogenicum) (PDB ID: 8HHL). Cas12f and Cas12m share high sequence similarity with TnpB proteins. Cas12f mol.2- and Cas12m-specific insertions (REC2) are highlighted in blue. These regions have a crucial role for the recognition of the PAM-distal region of the guide RNA–target DNA heteroduplex, suggesting that type V Cas12 enzymes acquired an ability to recognize the PAM-distal end of the guide RNA–target DNA heteroduplex in order to engage in CRISPR–Cas adaptive immunity. ZF, zinc-finger domain.

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Our structure also revealed that the ωRNA contains the guide–PK–stem structure, which is highly conserved among all guide RNAs of Cas12 enzymes (Extended Data Fig. 7a). This structural similarity strongly suggests that (1) the single guides of the tracrRNA-independent Cas12 variants (Cas12m to Cas12a) may have evolved from a condensed and duplicated version of the ωRNA ancestor to allow the formation of a CRISPR array (with palindromic repeats), and (2) the tracrRNA–crRNA pair present in some Cas12 systems (Cas12f to Cas12b) may have evolved from a split version of the ωRNA ancestor to allow the formation of a standalone tracrRNA gene, and a CRISPR RNA that later expanded into a full array (with non-palindromic repeats). A similar splitting hypothesis was proposed for the evolution of the Cas9 dual crRNA–tracrRNA guide from ωRNA2.

Methods

Sample preparation

The genes encoding ISDra2 TnpB (TnpB from D. radiodurans ISDra2; residues 1 to 408) and the 247-nt ωRNA were synthesized by Eurofin Genomics and cloned into the modified pETDuet vector (Novagen). The N-terminally MBP-tagged TnpB and ωRNA were co-expressed in Escherichia coli Rosetta2 (DE3). The E. coli cells were cultured at 37 °C until the A600 reached 0.8, and protein expression was then induced by the addition of 0.2 mM isopropyl β-d-thiogalactopyranoside (Nacalai Tesque). The E. coli cells were further cultured at 20 °C overnight, collected by centrifugation, resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, and 1 mM DTT), and then lysed by sonication. The lysates were centrifuged, and the supernatant was mixed with 3 ml of amylose resin (New England Biolabs). The mixture was loaded into a Poly-Prep column (Bio-Rad), and the TnpB–ωRNA complex was eluted with buffer B (20 mM Tris-HCl, pH 8.0, 40 mM maltose, 0.2 M NaCl, 5% glycerol, and 1 mM DTT). The complex was incubated with HRV3C protease overnight, and then loaded onto a 5 ml HiTrap Heparin column (GE Healthcare) equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, and 0.2 M NaCl). The peak fractions were collected and stored at −80 °C in buffer D (20 mM Tris-HCl, pH 8.0, 0.2 M NaCl and 20% glycerol) until use. Mutations were introduced by a PCR-based method, and sequences were confirmed by DNA sequencing (Supplementary Tables 1 and 2). Since the 3′ end of the tnpB gene overlapped with part of the ωRNA, it was difficult to perform PCR with the plasmid containing the full-length ωRNA. Thus, all TnpB mutants were created by introducing mutations on the DNA plasmid containing ωRNA with deleted 5′ region (−231 to −117).

Cryo-EM analysis

The TnpB–ωRNA–target DNA ternary complex was prepared for cryo-EM analysis according to the following procedure. A double-stranded DNA with phosphorothioate modifications at the cleavage sites was prepared by annealing a 35-nt target DNA strand and a 35-nt non-target strand containing a TTGAT TAM, at 95 °C for 2 min. The purified TnpB–ωRNA complex was incubated with the target DNA at room temperature for 30 min. The TnpB–ωRNA–target DNA ternary complex was purified on a Superdex 200 Increase 10/300 column (GE Healthcare), equilibrated with buffer E (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 10 μM ZnCl2, and 1 mM DTT). The purified complex solution (0.5 mg ml−1 final concentration) was applied to freshly glow-discharged Au 300 mesh R1.2/1.3 grids (Quantifoil) after adding 3 μl of amylamine, using a Vitrobot Mark IV (FEI) at 4 °C, with a waiting time of 10 s and a blotting time of 4 s under 100% humidity conditions. The grids were then plunge-frozen in liquid ethane cooled to the temperature of liquid nitrogen.

Cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Movies were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83Å, with a total dose of approximately 50 electrons per Å2 per 48 frames. The data were automatically acquired using the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −1.6 μm, and 3,570 movies were obtained.

Image processing

The data processing was performed with the cryoSPARC v3.3.2 software platform28. The dose-fractionated movies were aligned using patch motion correction, and the contrast transfer function (CTF) parameters were estimated using the Patch-based CTF estimation. From the 3,570 motion-corrected and dose-weighted micrographs, 2,136,853 particles were automatically picked using blob picker in cryoSPARC. The particles were subjected to several rounds of reference-free 2D classifications to create particle sets. The particles were curated by cryoSPARC heterogenous refinement (n = 4), using the map derived from the cryoSPARC ab initio reconstruction as a template. The selected particles were subjected to 3D variability analysis, and the resulting maps with different conformations were used for subsequent heterogeneous refinement. The best class containing 98,483 particles was refined using non-uniform refinement29 after CTF refinement, yielding a map at 3.21 Å resolution, according to the Fourier shell correlation (FSC) = 0.143 criterion30. The local resolution was estimated by cryoSPARC.

Model building and validation

The model was built using the predicted model of the ISDra2 TnpB protein created by AlphaFold2 as the reference31, followed by manual model building with COOT32. The model was refined using phenix.real_space_refine ver. 1.20.133, with secondary structure and metal coordination restraints. The metal coordination restraints were generated using ReadySet, as implemented in PHENIX. The structure validation was performed using MolProbity in the PHENIX package34. The EMRinger score35 and 3DFSC sphericity36 were calculated by PHENIX and the 3DFSC processing Server (https://3dfsc.salk.edu/upload/info/), respectively. The statistics of the 3D reconstruction and model refinement are summarized in Extended Data Table 1. The cryo-EM density map figures were generated using UCSF ChimeraX37. Molecular graphics figures were prepared using CueMol (http://www.cuemol.org).

Northern blotting analysis

Total RNA was extracted from cells expressing ωRNA or ωRNA-MBP or ωRNA-TnpB with TRIzol LS (Thermo Fisher Scientific) according to the manufacturer’s instructions. TnpB-interacting ωRNA was extracted from the purified TnpB–ωRNA complex with TRIzol LS (Thermo Fisher Scientific) according to the manufacturer’s instructions. Three micrograms of total RNA, 140 ng of in vitro-transcribed ωRNA and 140 ng of TnpB-interacting ωRNA were resolved by electrophoresis on a 10% polyacrylamide gel containing 7 M urea, followed by staining with GelGreen (Biotium). Fluorescence was visualized by an FLA-7000 imaging analyser (Fujifilm). The RNAs were transferred to a Hybond N+ membrane (Cytiva) by electroblotting for 1 h at 1.5 mA cm−2 in 1× TBE using a Transblot Turbo (Bio-Rad), and crosslinked by two rounds of UV irradiation (254 nm, 120 mJ cm−2; CL-1000, UVP). The membrane was treated with hybridization buffer (5% PEG 6000 (w/v), 7.5% SDS (w/v), 0.5% casein (w/v), 1 mM EDTA (pH 8.0), and 282 mM sodium phosphate buffer (pH 7.4)) at 52 °C for 1 h, and then subjected to hybridization with 2 pmol of the 5′-32P-labelled DNA probes at 50 °C overnight. The sequences of DNA probes are as follows: 5′-TTCTTCACTTCGGGATTCTTGAATC-3′ (probe I), 5′-CGTCTCGGTCATGGGTTTCCCCACA-3′ (probe II), 5′-GTCTGAGATTCCCGCAGCCACCAAC-3′ (probe III), 5′-GCAGACCATTGCCCGCCGAAGCAGG-3′ (probe IV), 5′-GGGCGCCAAGGGACTCTTGAACCTC-3′ (probe V) (Supplementary Table 2). The membrane was washed four times with 2× SSC (0.3 M NaCl, 30 mM sodium citrate (pH 7.0)), dried, and exposed to an imaging plate. Radioactivity was visualized by using an FLA-7000 imaging analyser. Uncropped images are available in the Source Data file.

LC–MS analysis

Each band on the gel was cut into cubes smaller than 1 mm3, soaked in 150 μl elution buffer (3 M sodium acetate (pH 5.3), 1 mM EDTA (pH 8.0), and 0.1% SDS), and shaken for 2 h at 37 °C. The buffer was transferred to a new tube. The gel fragments were then shaken with another 150 μl of elution buffer at 37 °C overnight. The elution buffers were combined, and after glycogen addition, the RNA was recovered by ethanol precipitation. The RNA precipitate was dissolved in water and digested by RNase A (Thermo Fisher Scientific), and then analysed by LC–MS. RNA fragment analysis was performed with an UltiMate 3000 RSLCnano system coupled with an Orbitrap Eclipse Tribrid (Thermo Fisher Scientific). One picomole of the RNA digest was diluted with 10 mM triethylammonium acetate, and loaded on a trap column (Acclaim PepMap 100 C18, 100 μm ID × 20 mm, Thermo Fisher Scientific). RNA fragments were separated on an ODS column (HiQ sil C18W-3, 100 μm ID × 100 mm, Techno Alpha) at a 300 nl min−1 flow rate. Separation was started with 99% mobile phase A (0.4 M hexafluoroisopropanol in water) and 1% B (0.4 M hexafluoroisopropanol in 50% methanol) for 10 min, and then B % was increased to 60% by linear gradient over 32 min. Eluents were injected into the ESI source through a nanoESI emitter (LOTUS emitters, FOSSILIONTECH), and ions were scanned by MS in the negative polarity mode.

In vitro DNA cleavage assay

For the in vitro cleavage assay, the TnpB–ωRNA complexes (wild-type or mutants) were purified in a similar manner to that for the complex prepared for the cryo-EM analysis. Protein concentrations were measured using a Bradford Protein Assay Kit (TAKARA). The DNA cleavage activity of TnpB was measured by in vitro DNA cleavage assays. The TnpB–ωRNA complex (2 μl, final concentration 250 nM) was mixed with the 3-kb linearized plasmid target containing the 16-nt target sequence and the TTGAT TAM (8 μl, 100 ng) (Supplementary Table 1), and incubated at 37 or 50 °C for 30 min in 10 μl reaction buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM DTT, and 5% glycerol). The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (Shimadzu). In vitro cleavage experiments were performed at least three times.

Mammalian genome editing assays

Mammalian cell culture experiments were performed in the HEK293FT cell line, grown in Dulbecco’s modified Eagle’s medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher), supplemented with 1× penicillin–streptomycin (Thermo Fisher) and 10% fetal bovine serum (VWR Seradigm). All cells were maintained at confluency below 80%. All transfections were performed with Lipofectamine 3000 (Thermo Fisher) in 96-well plates, unless otherwise noted. Cells were plated at approximately 20,000 cells per well 16–20 h prior to transfection, to ensure 90% confluency at the time of transfection. To evaluate indel efficiencies, transfection plasmids were combined with OptiMEM I Reduced Serum Medium (Thermo Fisher) to a total volume of 20 µl per well. Separately, 18.8 µl of OptiMEM was combined with 1.2 µl of Lipofectamine 3000. The plasmid and Lipofectamine solutions were then combined, and 10 µl was pipetted onto each well. Genomic DNA was collected 96 h after transfection by removing the supernatant and resuspending each well in 50 μl of QuickExtract DNA Extraction Solution (Lucigen). Cells were lysed by cycling at 65 °C for 15 min, 68 °C for 15 min, and 95 °C for 10 min. A 3 μl portion of lysed cells was used as the input in each PCR reaction for deep sequencing, and indel frequencies were quantified by CRISPResso238. Genome-wide off-target analysis was performed using tagmentation-based tag integration site sequencing (TTISS) as described previously25, and sites identified by TTISS were subjected to quantification of indel frequencies in a separate experiment (Supplementary Tables 1 and 2).

Reporting summary

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