Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation

Domain: neuroscience
Published: 2026-01-28
Editorial outcome: major_revision

Abstract
CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12aKD2P) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12aKD2P that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12aWT) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the “lid”, which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH’s structural organization in these mechanistically similar enzymes.

Introduction
CRISPR-Cas systems are adaptive immune systems of bacteria and archaea that protect them from invading genomes1,2. While there are different types of CRISPR-Cas systems based on the type of nucleic acid target and protein composition, the fundamental principle is RNA-guided DNA/RNA targeting followed by cleavage of the invader genome3. Of the different types, Class 2 systems composed of single multi-domain proteins are widely used for gene editing, including gene therapy4,5. Cas9 (signature protein of the type II system) and Cas12a (signature protein of the type V-A system) are Class 2 proteins widely used for genome applications6,7. These proteins target DNA by recognizing a 20-nucleotide-long complementary region between the crRNA-guide and the target DNA. Both Cas9 and Cas12a use proto-spacer adjacent motif [PAM; a short DNA motif (2-8 nucleotides) flanking the 20-nucleotide region] present in the target to distinguish self versus non-self DNA3,8,9.

Cas9 and Cas12a have similar protein organization features with two lobes, a recognition (REC) lobe and a nuclease (NUC) lobe to perform the functions of RNA-DNA recognition and DNA cleavage, respectively10. The two lobes are bridged by a conserved arginine/lysine-rich helix, called the bridge helix (BH), and previous studies have shown the importance of BH in RNA-DNA binding and cleavage11–18. Each lobe has multiple domains, and in general, both Cas9 and Cas12a undergo conformational changes to accommodate the RNA-DNA hybrid in a groove between the two lobes, even though the exact conformational changes are different3. Step-by-step R-loop propagation enables both Cas9 and Cas12a to have conformational checkpoints along the conformational cascade towards achieving the pre-catalytic stage such that formation of the catalytically competent state is tightly related to a full RNA-DNA base pairing in the R-loop (17-nucleotides in Cas9 and 16-nucleotides in Cas12a)19–21.

Previous structural12,20–26, computational27,28, and Förster resonance energy transfer (FRET)16,17,20,29,30 studies covering different orthologs have provided in-depth information on the conformational activation process of Cas12a. The apo-form of Cas12a is elongated25 and it transforms into a compact and closed crab-claw shaped structure after binding to crRNA (binary form)23,26,31. After binding to the target DNA (ternary form)12,20,21,23–25, the closed structure opens up to accommodate the growing hybrid in between the two lobes16,30. Upon full R-loop formation, there is opening up of the RuvC catalytic pocket through the conversion of a loop form of the “lid” covering the active site to a helix form, which enables passage of single-stranded DNA (ssDNA) into the RuvC active site pocket for cleavage20–22,27.

Off-target DNA cleavage is a problem in gene editing and different protein engineering strategies are being used to create high-fidelity Cas proteins for genome applications. Previous studies from our lab have shown that the conserved BH of Cas9 and Cas12a can be used for rationale protein engineering to remove off-target DNA cleavage13,14,32,33. Specifically, we targeted a region of the BH that undergoes a loop-to-helical transition in response to RNA-DNA binding [based on comparing different structures of Cas9 (PDBs: 4OO811, 5F9R34, 4ZT035, 4CMP6) and Cas12a (PDBs: 6GTC20, 6GTG20, 5NFV23, 5MGA22, 5B4312, 5ID626, 6I1L24, 6I1K24 and 5XUS36)] and introduced prolines to disrupt the smooth helical transition. The resulting variants showed higher fidelity in DNA cleavage13,14,32,33.

In this work, we report five cryo-electron microscopy (cryo-EM) structures of a Francisella novicida Cas12a (FnoCas12a) variant with two proline substitutions in the BH (K969P/D970P; referred to as FnoCas12aKD2P). The structures unravel a mechanism by which the BH of Cas12a plays an integral part in the smooth transition of the different intermediate states to reach the final active conformation. Specifically, the loop-to-helical transition and the bending of BH enables the propagation of the RNA-DNA hybrid, which allosterically coordinates several downstream processes required to attain a cleavage-competent state, including the opening of the RuvC-lid. Biochemical and computational analyses show that BH’s helical transition is critical for the rest of the conformational changes, with a positively correlated movement of BH–RuvC-helix 1 (H1) movements with that of RuvC-lid, crRNA, and DNA. Interestingly, structural comparisons of the BH of Cas12 and Cas9 proteins show distinctions in the structural organization of BH and the RuvC-H1 implicating potential differences in the activation of these diverse, yet mechanistically similar, protein families. Since BH is conserved in several types of Cas proteins, as well as in other RNA-binding proteins such as RNA polymerases, our work provides molecular insights into the fidelity of DNA/RNA catalyzing enzymes.

Results
FnoCas12aKD2P samples different conformational states toward the DNA cleavage competent state
To assess how the conserved BH of Cas12a and its helical transitions promote conformational changes needed for DNA cleavage, we determined cryo-EM structures of FnoCas12aKD2P. We reconstituted FnoCas12aKD2P with its crRNA and a 24-nucleotide-long double-stranded DNA (dsDNA) substrate where the target strand (TS) will base pair (bp) with the guide region of the crRNA and the non-target strand (NTS) will exist as ssDNA in the R-loop. The DNA possesses 4-nucleotides of PAM and 20-nucleotides of protospacer, that is numbered from 0 to 19 in the PDB files. The ternary complex was assembled in the absence of a divalent metal to prevent DNA cleavage and purified over size exclusion chromatography (SEC). The peak corresponding to the ternary complex, peak 2 (Supplementary Fig. 1), was used to prepare cryo-grids. We consistently got better distribution of the particles when we used graphene oxide-coated grids (Supplementary Method 1, Supplementary Fig. 2)37,38. A 3D map with ~2.96 Å resolution was created from ~400K particles and the map possessed the typical crab-claw shape similar to the wild-type (FnoCas12aWT, PDB: 6GTG20). Further 3D classification generated six subclasses with an almost equal distribution of particles (14% to 18%, Supplementary Fig. 3), five of which ranged in resolution from 3.2 Å to 4 Å and were used for model building and analysis (Supplementary Fig. 4, Supplementary Table 1, and Supplementary Method 2). These five structures have differences in domain placements and represent conformational transitions needed to activate Cas12a for DNA cleavage (Fig. 1). The maps and models of FnoCas12aKD2P ternary complex from this study were compared with those of FnoCas12aWT from previous studies (Supplementary Table 2), including one that used single-molecule FRET (smFRET) analysis to identify the different conformational states (intermediate states I1-I5) towards attaining the pre-catalytic conformation20. Additionally, we compared FnoCas12aKD2P structures with Acidaminococcus sp. (As) Cas12a structure-series that captured different conformations at different lengths of the R-loop formation21. We named the different states of the FnoCas12aKD2P complex as state 1 (S1) to state 4b (S4b) (Fig. 1b–f). These structures provide mechanistic information about the role of BH in orchestrating the conformational cascade to activate Cas12a for DNA cleavage, as detailed below, including the opening of the lid covering the RuvC active site through direct interactions with the BH residues.

Comparison of the map features of all five states revealed that the major difference between them is in the movements of the contiguous REC1 and REC2 domains (Supplementary Movie 1) and visibility of REC2, which were previously shown to be important to open the closed binary complex of FnoCas12aWT to accommodate the RNA-DNA hybrid in between20,23,26. Notably, in state FnoCas12aKD2P-S1, REC2 lies in close contact with the Nuc domain, giving it a closed conformation (Fig. 1g and Supplementary Table 3), and the PAM-interacting (PI) domain remains open compared to the other states due to some disordered residues. Additionally, FnoCas12aKD2P-S1 does not show a continuous stretch of density for DNA, but there is residual density for positions 4 to 6 of the TS DNA (Supplementary Fig. 5a, b). An analysis of the seed region of the crRNA shows that there is preordering of this region similar to the binary complex (PDB: 5NG623, Supplementary Fig. 5c, d), indicating that the BH substitutions do not impact binary complex formation. These factors suggest that the FnoCas12aKD2P-S1 state represents initiation of the ternary complex formation, where the complex is in very early stages of PAM recognition.

In FnoCas12aKD2P-S2, the ternary complex transitions toward R-loop formation. The protein undergoes a closed-to-open transition, where the REC1 and REC2 domains begin to move away from and upward relative to the NUC lobe to accommodate the R-loop (Fig. 1h, Supplementary Movie 1, Supplementary Tables 3 and 4). During the transition of S1 to S2, the REC1 domain undergoes ~14° rotation with respect to a horizontal axis passing through the PI and Nuc domains (referred to as NUC-lobe axis hereafter, Supplementary Figs. 6 and 7a) and a translation of ~14 Å (distance between PI and REC1 domains). In contrast, the REC2 undergoes only a minor rotation of ~1° away from the NUC-lobe axis and 1 Å away from the Nuc domain, compared to that in S1 (Supplementary Fig. 7a, Supplementary Tables 5a, b). We observe 8-nucleotides of base pairing between crRNA and TS DNA in FnoCas12aKD2P-S2 (Supplementary Fig. 8a, b and Supplementary Table 6). However, due to the disordered PI domain, neither the density for the PAM sequence nor the rest of the NTS is visible. FnoCas12aKD2P-S2 resembles the FnoCas12aWT-I2 structure (PDB: 6GTD20; RMSD 3.6 Å, Supplementary Table 2).

In FnoCas12aKD2P-S3, the movements of the REC1 and REC2 domains increase the accessibility for the RNA-DNA hybrid (Supplementary Fig. 7b). The movement of the REC1 domain in FnoCas12aKD2P-S3 is similar to that observed in FnoCas12aWT-I2 and FnoCas12aWT-I3 states20 (Supplementary Table 5a). We observe a complete absence of the density for the REC2 domain in FnoCas12aKD2P-S3, and hence this domain was omitted from the model (Fig. 1d, i). While the REC2 domain was flexible in the FnoCas12aWT structures, none of them were captured with a complete absence of density for this domain. Similar states with a dislodged REC2 without density were captured for AsCas12a with 8- (PDB: 8SFI21) and 10- (PDB: 8SFJ21) bp of RNA–TS DNA hybrid during R-loop propagation, pointing to large movements needed for REC2 to accommodate the growing R-loop. In FnoCas12aKD2P-S3, 8-nucleotides, out of the total 15 nucleotides that are visible for the TS, are base-paired with the crRNA-guide region (3-nucleotides at the end of R-loop shows distorted pairing), and 3-nucleotides out of 7-nucleotides visible for the NTS exist as a single strand (Supplementary Fig. 8c and Supplementary Table 6). It is interesting to note that the PI domain is not ordered in FnoCas12aKD2P-S2 with 8-nucleotides of base pairing, but it is in FnoCas12aKD2P-S3 with similar base pairing and a visible NTS. This may correlate with the target DNA search mechanism of Cas12a, where establishing a critical amount of base pairing between the TS and crRNA-guide and passage of NTS into the hybrid channel may trigger PI ordering as a cue to further proceed with R-loop formation rather than dissociation and search for another potential DNA target39. The lysine helix-loop (LKL, residue 662-679) that was shown to initiate PAM unzipping in FnoCas12aWT (PDB: 5MGA22) is observed in FnoCas12aKD2P-S3 with lysine(s) interacting with the PAM nucleotides (Supplementary Fig. 9). However, these interactions are not observed in FnoCas12aKD2P-S1 and FnoCas12aKD2P-S2 due to the disordered PI domain. The FnoCas12aKD2P-S3 appear to be an intermediate state between FnoCas12aWT-I2 (3.6 Å RMSD between S3 and PDB: 6GTD20) and FnoCas12aWT-I3 (2.5 Å RMSD between S3 and PDB: 6GTE20) (Supplementary Table 2).

The remaining two states of the FnoCas12aKD2P structures are very close in conformations without significant differences to place them in order towards reaching the active state. These states closely resemble the intermediate state FnoCas12aWT-I320 (S4a: 5.2 Å RMSD and S4b: 4.3 Å RMSD, Supplementary Table 2 and Supplementary Fig. 10) and the two intermediate states of AsCas12a with 15- and 16-bp of R-loop formation (PDB: 8SFL and 8SFN21, respectively, Supplementary Table 2). In FnoCas12aKD2P-S4a, we see less well-defined density for REC2 (similar to the 15-bp complex of AsCas12a), with a density pattern showing stretched features consistent with the high flexibility of this region. With the current positioning of REC2 in FnoCas12aKD2P-S4a, we observe a rotation of ~17° upwards of the NUC-lobe axis and a translation of ~14 Å away from the Nuc domain, compared to that in FnoCas12aKD2P-S2 (Supplementary Fig. 7c and Supplementary Table 5b), with the distance between REC1 and REC2 increasing to ~77 Å (Fig. 1j and Supplementary Table 4). FnoCas12aKD2P-S4a has the most visible length of RNA-DNA hybrid among all five states, with 11 bps of hybrid between crRNA and TS DNA (4-nucleotides at the end of R-loop show distorted pairing) and 4-nucleotides of the single-stranded NTS (Supplementary Fig. 8d and Supplementary Table 6). FnoCas12aKD2P-S4b has the highest resolution (3.21 Å) among all five states and has clear density for the REC2 domain (Fig. 1f, k). The REC2 domain rotates downwards to the central cavity compared to its position in S4a (Supplementary Tables 3 and 5b, Fig. 1k and Supplementary Fig. 7d, e). In S4b, we see 9-bp of crRNA:TS DNA hybrid (5-nucleotides at the end of R-loop distorted) and 7-nucleotides of NTS DNA (Supplementary Fig. 8e and Supplementary Table 6).

Overall, comparing all five states of FnoCas12aKD2P, we observe that the maximum movement of the REC1 domain is the 14 Å translation between PI and REC1 domains between states S1 and S2, with a stable placement of REC1 and Nuc domains across all the states (Supplementary Table 5a, Supplementary Figs. 7 and 10). Comparatively, REC2 moves to a maximum of 34 Å in S4a relative to S1 and then moves closer (by 15 Å) to the NUC-lobe axis in S4b (Supplementary Table 5b, Fig. 1g–k distance indicated by solid line and Supplementary Fig. 7f). These movements increase the distance between the REC1 and REC2 domains (~35 Å between S1 and S4 states, Supplementary Table 4) as the system advances towards the pre-catalytic state (Fig. 1g–k distance indicated by dashed line, Supplementary Figs. 7 and 10). As the REC lobe opens, the R-loop grows in-between the two lobes.

Sampling of the pre-catalytic state is reduced in FnoCas12aKD2P
We compared the conformational states of FnoCas12aKD2P with those of the FnoCas12aWT from different studies (Supplementary Table 2). FnoCas12aKD2P-S1 appears to be a conformation between the FnoCas12aWT-binary23 and the FnoCas12aWT-I1 (PDB: 6GTC20), which is the initial stage of ternary complex formation based on smFRET studies (Supplementary Fig. 10). FnoCas12aKD2P-S2 is similar to FnoCas12aWT-I2 due to the similar positions of REC1 and REC2 domains (Supplementary Table 4). There is a ~7° rotation of REC2 towards the NUC-lobe axis in FnoCas12aWT-I4 compared to that in FnoCas12aWT-I320 (Supplementary Table 5b). The additional states of FnoCas12aKD2P-S3 and FnoCas12aKD2P-S4a likely represent intermediate states between FnoCas12aWT-I2 and FnoCas12aWT-I3 that were captured during this rotation of REC2, supported by the weak density for REC2 in these states. While there is ordering of the REC2 density in state FnoCas12aKD2P-S4b, only 9-nucleotides of base pairing with the crRNA guide are visible, compared to 11-nucleotides of base pairing in FnoCas12aKD2P-S4a (Supplementary Fig. 8d, e and Supplementary Table 6). Overall, S4a and S4b of FnoCas12aKD2P appear to be very transient states towards attaining a pre-catalytic state (FnoCas12aWT-I4)20 (Supplementary Fig. 10). While we captured all the different states with an equal probability (Supplementary Fig. 3f), we did not observe a pre-catalytic state where the RNA-DNA hybrid is ordered, along with more visibility for NTS closer to the RuvC active site pocket, and also with the critical loop-to-helical transition in of the lid region of the RuvC domain (RuvC-lid) to open the catalytic pocket. An impaired BH due to the double proline substitutions appears to reduce the efficiency of conformational transitions to reach the catalytically competent state, which also enabled capturing of sub-intermediate states that were to the best of our knowledge not previously observed. This observation supports the lower rate constant that was observed for DNA cleavage by FnoCas12aKD2P compared to that by FnoCas12aWT (3-fold reduction in on-target DNA cleavage)14.

Impaired BH restricts conformational checkpoints during R-loop formation
Two regions present in the REC1 domain, REC-linker (324-331) and finger (301-309), were shown to sense the RNA-DNA hybrid formation through PAM-proximal and PAM-distal interactions, respectively20. The movement of the REC-linker across the states in FnoCas12aKD2P remains similar to that in FnoCas12aWT (Supplementary Fig. 11). In FnoCas12aKD2P-S1, the REC-linker is positioned away from the crRNA, similar to the R-loop initialization complex of FnoCas12aWT (PDB: 6GTC20) and subsequently shifts closer to the crRNA in states S2 to S4b of FnoCas12aKD2P (G5 to C7, PAM-proximal region, similar to that in FnoCas12aWT-I4, PDB: 6GTG20), with a concomitant R-loop propagation in the later states (Supplementary Fig. 11 and Supplementary Table 7). The finger region moves closer to the PAM-distal region of the RNA-DNA hybrid during the transition of FnoCas12aKD2P from states S1 to S4b. Despite this movement, docking of the finger region onto the crRNA at nucleotide positions 15–17, which was identified as a delayed checkpoint towards the catalytic activation of Cas12a20,22, is not observed in the structural transitions that were captured for FnoCas12aKD2P. Overall, while the interaction of the REC-linker at the PAM-proximal side is intact in FnoCas12aKD2P, the interaction of the finger region at the PAM-distal side is impaired.

A comparison of the RNA-DNA hybrid in different states of FnoCas12aWT and FnoCas12aKD2P shows that the BH is essential for RNA-DNA hybrid propagation and proper base pairing. An analysis of the distance evolution between the BH and the RNA-DNA hybrid is shown in Supplementary Table 8. K969 is at a distance of 34 Å from U4 of the crRNA in the FnoCas12aWT-binary structure (PDB: 5NG6)23, and the corresponding distance is 34 Å between P969 and A4 in FnoCas12aKD2P-S1, showing similar positioning of the crRNA in these two states (Supplementary Table 8). In the pre-catalytic state of FnoCas12aWT-I4, the distance between the RNA-DNA hybrid gets closer to K969 (~6 Å with U11) when the R-loop has a full 20-nucleotides of RNA-DNA base pairing20 (Fig. 2a, b, and Supplementary Table 8). In FnoCas12aKD2P-S4a and FnoCas12aKD2P-S4b, the RNA-DNA hybrid remains farther (20 Å and 27 Å, respectively) from the BH due to the inability of the BH to undergo the loop-to-helical transition, which prevents its bending towards the hybrid (Fig. 2a, c, Supplementary Fig. 12 and Supplementary Table 8).

In FnoCas12aKD2P states, RNA-TS DNA base pairing pattern is impacted. In FnoCas12aKD2P-S2, a region of the PI domain (S695-I714) is disordered, and there is no clear density for the PAM sequence of the DNA compared to clear visibility of 4-nucleotides of PAM and 9-nucleotides of RNA-DNA hybrid in FnoCas12aWT-I120. In FnoCas12aKD2P-S2 and FnoCas12aKD2P-S3, after dG(−7), the R-loop exhibits unstable base pairing. However, for FnoCas12aKD2P-S4a and FnoCas12aKD2P-S4b, base pairing is stable until position dA(−11) and dA(−10) of TS DNA, respectively, with the corresponding complementary position of the crRNA-guide, and the remaining visible nucleotides (4 in FnoCas12aKD2P-S4a and 3 in FnoCas12aKD2P-S4b) being in a single stranded condition (Supplementary Fig. 8). We observe stacking and distortion of the single-stranded form of the crRNA and the TS DNA in FnoCas12aKD2P structures, beyond the base paired region. This indicates that the BH conformational change is essential for progressive R-loop formation, stable base pairing of the RNA-DNA hybrid, and to enable helix formation of the RNA-DNA hybrid.

Cooperativity in the conformational transitions of BH and RuvC-helix 1 is critical for Cas12a’s activity
It was previously reported that BH and RuvC-helix 1 (RuvC-H1) of RuvC motif-II undergo coordinated conformational transitions while transforming from the binary to the ternary complex16,17,21. A comparison of the 8-bp R-loop state (FnoCas12aWT-I3)20 and the 20-bp R-loop state (FnoCas12aWT-I4)20 of the ternary complex structures show that there is melting of RuvC-H1 upon partial R-loop formation in state I3, followed by elongation of the BH by 3 turns in state I420. In addition, the BH-end (D970) bends by 28° and moves ~20 Å towards the RNA-DNA hybrid after full R-loop formation compared to the position in the partial R-loop structures (Fig. 2a–c, Supplementary Fig. 12, and Supplementary Tables 8 and 9). Comparing the structure of Cas12a’s BH in the different states of FnoCas12aKD2P in our study with that of wild-type, we find that, while the RuvC-H1 melting is unaffected, the loop-to-helical transition of the BH is impaired due to the proline substitutions. The impairments in the elongation and bending of the BH keep it positioned at a distance of ~20 Å away from the RNA-DNA hybrid in both S4a and S4b of FnoCas12aKD2P (compared to ~17 Å in FnoCas12aWT-I1 and ~6 Å in FnoCas12aWT-I420, Supplementary Table 8).

Another important conformational change towards activation of Cas12a is the insertion of the conserved W971 (residue immediately following the proline substitutions of FnoCas12aKD2P at 969 and 970) into the REC2 hydrophobic pocket (composed of amino acids Y579, K527, K524, R583 of FnoCas12aWT12,16,22) to enable REC2 docking after hybrid formation. In FnoCas12aKD2P structures, we see insertion of W971 into the REC2 hydrophobic pocket in S1 and S4b (S2 and S4a do not show density in this area to determine side chain position). However, the interacting distance of W971 with Y579 of the REC2 domain, which is at ~4 Å in FnoCas12aWT-I4, remains farther in S1 and S4b of FnoCas12aKD2P (6 Å and 10 Å, respectively) (Supplementary Fig. 13). It can be envisioned that W971 will be sturdier in a completely elongated BH than when present in an impaired BH. These observations indicate that impairing the loop-to-helical transitioning of BH potentially prevents sampling of the activated state in our cryo-EM classes due to the absence of an elongated and bent BH to latch on to the RNA-DNA hybrid and due to the potential hindrance in coordinating the distinct REC2 movements across the different states through locking of W971 with the REC2 domain.

BH is an allosteric regulator that connects RNA-DNA hybrid formation to the opening of the lid of the RuvC active site
Previous studies have shown that a region called the RuvC-lid (residues 1009 to 1021 in FnoCas12a) occludes the active site pocket of the RuvC during the earlier states of the conformational cycle by existing as a loop. To attain the pre-catalytic state, the RuvC-lid undergoes a loop-to-helical transition, exposing the RuvC active site for DNA access20,21,24. A comparison of the different states of FnoCas12aKD2P with that of FnoCas12aWT provides interesting insights into the cooperative action of the BH and the RuvC-lid to open the RuvC active site for DNA access.

Compared to the conformation in the binary FnoCas12aWT structure23, region comprising E961-D970 of the BH undergoes loop-to-helical transition in response to full R-loop formation in the ternary complex20,23. Similar to AsCas12a, in FnoCas12aWT, the residue E1020 in the RuvC-lid forms an electrostatic interaction with K981 in RuvC-H1, stabilizing the loop form of the RuvC-lid and occluding the RuvC active site in partial R-loop states (until ~15-bp of R-loop formation as observed in AsCas12a)20,21,27. This interaction is disrupted in the pre-catalytic state in FnoCas12aWT-I4 with the concomitant formation of additional interactions between BH and RuvC-lid (R964/R968-E1020) and RuvC-H1 and RuvC-lid (K981-K1021), thus opening the RuvC active site to ssDNA20,21,31 (Fig. 3a–d, Supplementary Figs. 14, 15 and Supplementary Table 10). To understand the importance of these interactions in Cas12a’s activation, we generated alanine substituents of R964, E1020, K1021, and E1020A/K1021A in both FnoCas12aWT and FnoCas12aKD2P backgrounds. R968 was not substituted due to its documented role in binary complex assembly and loss of activity upon alanine substitution7. Recombinantly purified proteins (Supplementary Fig. 16) were used to perform plasmid cleavage assays with different substrates: matched DNA (MCH, complete complementarity with the guide region of the crRNA) and with two different DNA substrates with mismatches (MM2: mismatch at position 2 proximal to the PAM and MM13, mismatch at position 13 from the PAM) (Fig. 3e, f, Supplementary Fig. 17).

All FnoCas12aWT variants (E1020A, K1021A, R964A, E1020-K1021A) retain high linearization activity (≥90% at 15 min) that is comparable to wild-type for MCH substrate. In the FnoCas12aKD2P background, E1020A and K1021A variants show similar linearization (~85% at 15 min) to FnoCas12aKD2P–MCH, which has a slower rate compared to FnoCas12aWT–MCH substrate. FnoCas12aKD2P-E1020A/K1021A shows reduced linearization (47% at 15 min), with a 2-fold and 1.8-fold decrease compared to FnoCas12aWT–MCH and FnoCas12aKD2P–MCH, respectively (Fig. 3e, Supplementary Fig. 17a and Supplementary Table 11a). FnoCas12aKD2P-R964A displays a pronounced loss of linearization (12% at 15 min), representing an 8-fold decrease relative to FnoCas12aWT–MCH and a 7-fold decrease relative to FnoCas12aKD2P–MCH. Total cleavage is also low for FnoCas12aKD2P-E1020A/K1021A (68% at 15 min) and FnoCas12aKD2P-R964A (38% at 15 min), with the latter accumulating 26% nicked intermediate (Supplementary Fig. 18a and Supplementary Table 11a). These results indicate that in the absence of a BH loop-to-helical transition, R964A and E1020A/K1021A substitutions in FnoCas12aKD2P variants reduce the efficiency of DNA cleavage, with a more profound effect in R964A. Additionally, it also shows that in FnoCas12aWT, which possesses an intact transitioning of the BH, disrupting the BH–RuvC-lid interactions do not critically impact on-target DNA cleavage.

Similar to FnoCas12aWT–MCH condition, all FnoCas12aWT variants (E1020A, K1021A, R964A, E1020-K1021A) maintain efficient linearization of MM2 substrate (82-90% at 15 min). FnoCas12aKD2P-E1020A and FnoCas12aKD2P-K1021A show moderately reduced linearization (62-70% at 15 min) relative to FnoCas12aWT–MM2 but are similar to FnoCas12aKD2P–MM2 (65% at 15 min). FnoCas12aKD2P-E1020A/K1021A has a reduced linearization (40% at 15 min) and an accumulation of nicked intermediate (38%) compared to FnoCas12aKD2P–MM2 (23% nicking, 65% linearization at 15 min) (Supplementary Figs. 17b, 18b and Supplementary Table 11b). FnoCas12aKD2P-R964A–MM2 exhibits severely impaired linearization (4%), with 22-fold and 16-fold reductions relative to FnoCas12aWT–MM2 and FnoCas12aKD2P–MM2, respectively, with total cleavage at 45% (41% nicking at 15 min) (Supplementary Fig. 18a, b and Supplementary Table 11b). These data suggest that combining FnoCas12aKD2P with R964A or E1020A/K1021A enhances sensitivity to PAM-proximal mismatches.

With an MM13 mismatch, FnoCas12aWT variants E1020A, K1021A, and E1020A/K1021A retain linearization comparable to FnoCas12aWT–MM13 (84-90% at 15 min), but FnoCas12aWT-R964A shows a sharp reduction (12%, 7.5-fold lower than FnoCas12aWT–MM13) with 78% nicked intermediate accumulation, yielding a total cleavage similar to FnoCas12aWT–MM13 (~90%). FnoCas12aKD2P-E1020A and FnoCas12aKD2P-K1021A, similar to FnoCas12aKD2P, exhibit poor linearization of MM13 (0–5% at 15 min). This represents 29-fold and 43-fold reduction, respectively, for these variants, compared to their efficiency to linearize MCH, and 21-fold and 35-fold reduction, respectively, in their efficiency to linearize MM2 (Fig. 3f, Supplementary Fig. 17c and Supplementary Table 11c). Similar to FnoCas12aKD2P–MM13, these variants accumulated high nicked intermediate (~85%), with a total cleavage of 88–90% at 15 min. Notably, FnoCas12aKD2P-R964A and FnoCas12aKD2P-E1020A/K1021A are defective in both strand cleavages, with 11% and 40% nicked intermediates, and linearization limited to 1% and 0%, respectively, at 15 min (Supplementary Fig. 18a and Supplementary Table 11c). These data indicate that R964A severely impairs second-strand cleavage in the FnoCas12aWT background for mid-R-loop mismatches. However, in the FnoCas12aKD2P background, R964A and E1020A/K1021A substitutions impair cleavage of both DNA strands in all substrates that were tested, including on-target and off-target substrates.

Our findings show that contacts between the BH and RuvC-lid (R964-E1020, E961-K1021) are only secondary to the BH’s helical transition for orchestrating the conformational transitions required for double-strand cleavage and mismatch sensitivity. The wild-type protein with an intact conformational change in the BH can linearize (i.e., both NTS and TS cleavages) despite disrupting the BH–RuvC-lid interactions, with the only significant reduction being in the second strand cleavage for MM13 substrate (Supplementary Figs. 17, 18a, and Supplementary Table 11). On the contrary, when BH’s sturdiness is restricted, as in the FnoCas12aKD2P variant, the enzyme becomes highly dependent on these interactions to provide RuvC active site access to ssDNA. In the FnoCas12aKD2P background, while R964A and E1020A/K1021A substitutions impair both strand cleavages, single substitutions (E1020A or K1021A) impede second-strand cleavage (Supplementary Fig. 18a and Supplementary Table 11). These results demonstrate that the BH’s loop-to-helical transition is an allosteric switch coupling R-loop propagation to catalysis and point to the importance of the helicity of the BH in assisting the passage of TS DNA into the RuvC active site pocket. BH, RuvC-H1, and RuvC-lid are potential engineering sites to reduce mismatch tolerance in Cas12a.

Impaired loop-to-helical transition of the BH creates conformational states that are distinct from those of the wild-type protein
Using Molecular Dynamics (MD) simulations and principal component analysis (PCA), we explored the similarities and differences of the conformations of states S2 and S4b of FnoCas12aKD2P with state FnoCas12aWT-I420 (Supplementary Method 3 and Supplementary Fig. 19). The resulting 2D projection of first and second principal components (PC1 and PC2, respectively, Supplementary Movies 2 and 3) revealed distinct clustering for each state, indicating well-defined conformational ensembles (Fig. 4). Furthermore, along PC1 (79.2%), a progression is observed from S2, through S4b, to finally attain the I4 conformation, delineating a pathway consistent with the expected structural transitions toward the pre-catalytic state of Cas12a (Fig. 4a). Also, these MD simulations support the notion that FnoCas12aKD2P-S2 and FnoCas12aWT-I220 states are closely related, while FnoCas12aKD2P-S4b represents a distinct intermediate conformation between FnoCas12aWT-I2 and FnoCas12aWT-I4 (Supplementary Fig. 20).

An analysis of the structural differences of the region comprising the BH, RuvC-H1, and RuvC-lid shows the main differences in conformation contributing to PC1, representing 79.2% of the total variance (Fig. 4b and Supplementary Movie 2). In FnoCas12aKD2P-S2, BH is shorter, RuvC-H1 is longer, and RuvC-lid is in the loop form. In FnoCas12aWT-I420, all features of the active state are present: elongated and bent BH, melted RuvC-H1, and a helical RuvC-lid. FnoCas12aKD2P-S4b shows features that are intermediate to FnoCas12aKD2P-S2 and FnoCas12aWT-I420 states. In FnoCas12aKD2P-S4b, BH is longer than that in FnoCas12aKD2P-S2, RuvC-H1 has started to melt, while the RuvC-lid is still in the loop form. The PC2 captures complementary motions of the same regions but accounts for only 16.6% of the total variance (Supplementary Movie 3). These results show that coordinated and complete transitions of the BH and RuvC-H1 are a prerequisite for RuvC-lid’s helical transition. The complete analysis, including state FnoCas12aWT-I220 that lack side chains in the experimental structure, is available in the SI (Supplementary Method 3 and Supplementary Figs. 19, 20).

Loop-to-helical transition of the BH enables a coordinated communication between different regions of Cas12a and nucleic acids
Our structures reveal differences in the interaction of Cas12a with crRNA and DNA at different states (Fig. 4c–e and Supplementary Fig. 21). To delineate potential allosteric communication between BH and nucleic acid, we performed correlation analyses of residues E961, R964, R968, K981, E1020, K1021, and N1022 of the BH, RuvC-H1, and RuvC-lid, as well as nucleotides in crRNA and DNA (Fig. 4c–e). The complete correlation analysis is available in Supplementary Fig. 21 and Supplementary Tables 12–20. Results reveal that BH–RuvC-H1 and RuvC-lid conformations exhibit strongly correlated motions with those of the crRNA and DNA, and that the motions are different between the different states. FnoCas12aKD2P-S2 has fewer correlated motions (i.e., components move in the same direction) between the BH–RuvC-H1 and RuvC-lid, while there are several anti-correlated motions (i.e., components move in the opposite direction) between BH–RuvC-H1 and the RNA and DNA (Fig. 4c and Supplementary Tables 13, 16, and 19). In the intermediate state FnoCas12aKD2P-S4b, however, this correlation pattern shifts; there is an increase in the correlated motions between BH–RuvC-H1 and RuvC-lid and a few additional correlated motions with the crRNA, while there still is a substantial number of anti-correlated motions with crRNA and DNA (Fig. 4d and Supplementary Tables 13, 16, and 19). In FnoCas12aWT-I420, we see only correlated motions between BH–RuvC-H1, RuvC-lid, crRNA, and DNA (Fig. 4e and Supplementary Tables 14, 17, and 20). Interestingly, some of the correlated motions with crRNA and DNA include nucleotides near the middle of the R-loop (~8) providing mechanistic clues on why mismatches between the crRNA and DNA in the R-loop away from the PAM are particularly sensitive in FnoCas12a. This reorganization of the correlation network in different states of the protein points to the pivotal role that the BH–RuvC-H1 transitions play in organizing the cleavage-competent form of Cas12a.

Helix 1 from the RuvC motif-II of the Cas12 protein family is adopted for coordinated conformational changes with the BH
The coordinated conformational change of BH with the first helix of the RuvC motif-II (RuvC-H1) in Cas12a’s activity was previously proposed17, and its structural mechanisms are revealed in FnoCas12aKD2P structures. To further understand the importance of the coordination of BH and RuvC-H1 and its conservation in other Cas family proteins, an HMM (Hidden Markov Model) profile of these two helices was used to search the HMMER40 database. The result showed hits only from the Cas12a subtype, indicating that the sequence conservation around this region is limited to only Cas12a orthologs (Supplementary Fig. 22).

We next analyzed whether there is structural conservation of these two helices in Cas12 subtypes. We used BH and RuvC domain (motifs I, II, and III) of FnoCas12a (PDB: 5NFV23) as the query in DALI (Distance Matrix Alignment) server41. The structural analysis identified similar structural folds in other Cas12 subtypes, resolvase, and TnpB. This is consistent with previous studies that indicated TnpB as the ancestor for Cas12 proteins42 with conserved structural similarities43. Since Cas9 also has a BH and a RuvC domain (composed of motifs I, II, and III), we included selected Cas9 subtypes along with the results from the DALI output and performed an “all-against-all structure comparison” using the DALI server. A dendrogram based on this analysis groups all the proteins into five clusters, with the Cas9 subtypes occupying a distinct cluster and the Cas12 subtypes grouping into different sub-clusters (Fig. 5a). These observations show that there are some inherent structural differences between Cas12a and Cas9, which may translate to differences in the conformational activation processes. Cas12 structural analysis shows that the relative orientation of BH and RuvC-H1 is similar and that RuvC-H1 overlays very well in all the Cas12 subtypes. Comparing the BH between the Cas12 subtypes, the length and orientation of BH are similar in Cas12e44, Cas12f45, Cas12j46,47, Cas12k48, Cas12g49, Cas12c150, Cas12i151, and Cas12i252 (Fig. 5b and Supplementary Fig. 23). Variations are observed in the BH of Cas12j46,47, Cas12l53, Cas12f45, and Cas12i151 since their BHs possess an insertion domain or have folded double helices compared to only a loop in FnoCas12a (Fig. 5 and Supplementary Fig. 23).

Structural overlays of the BH and RuvC domain (motifs I, II, and III) of Cas9 (subtypes II-A, II-B, and II-C) with those of FnoCas12a show that the BH and a helix of the RuvC motif-II of Cas9 have a similar structural arrangement to that in Cas12a. While helix in the RuvC motif-II of Cas9 and Cas12a RuvC-H1 shows a better structural overlay, the BH of Cas9 is prominently longer and does not align with that of FnoCas12a (Fig. 5b, Supplementary Figs. 23, 24). Another significant difference between Cas9 and Cas12 is the insertion of the REC lobe between BH and RuvC motif-II in Cas9, compared to their direct connection in Cas12 families (Supplementary Fig. 24). This long separation can potentially disrupt coordinated conformational changes between BH and RuvC-H1 that are present in Cas12s. Within the Cas9, there is a loop-to-helical transition of the BH in response to RNA binding that has been demonstrated to impact DNA cleavage efficiency and DNA mismatch sensitivity6,11,13,32,33,35. These observations show that while the fundamental mechanism of loop-to-helical transition of the BH is conserved in Cas12a and Cas9, the mechanisms for coordinated conformational changes may differ between them. While the RuvC-H1 of RuvC motif-II is co-opted by Cas12 families to undergo coupled transformations with the BH to orchestrate conformational changes needed for assembling the pre-catalytic state, such coupling is absent in Cas9 (Supplementary Fig. 24c). Thus, nature seems to have optimized Cas12a and Cas9 molecular mechanisms differently to attain the same goal of RNA-directed, sequence-specific, DNA cleavage.

Methods
Plasmid preparation and mutagenesis
The Francisella novicida (Fno) cas12a wild-type and K969P-D970P variant (cas12aKD2P) genes that were cloned in a pET28a-based vector with an N-terminal Maltose Binding Protein (MBP) tag were used as backbones for generating all the protein variants9,14. Variants were constructed by site-directed mutagenesis using the primers listed in Supplementary Table 21. Resultant PCR products were transformed into Escherichia coli DH5α cells, and selected clones for each variant were sequence confirmed by nanopore sequencing (whole plasmid sequence for all the clones are available in Source Data). One sequence-confirmed clone for each variant was subsequently transformed into E. coli Rosetta (DE3) cells for protein overexpression.

Protein expression and purification
The plasmids containing the Fno cas12a gene with the different substitutions were transformed into E. coli Rosetta (DE3) cells and purified using established procedures9,14,56. Briefly, Rosetta cells with the expression plasmid were grown in 2x Yeast Extract Tryptone (2xYT) medium supplemented with 50 μg mL−1 Kanamycin and 34 μg mL−1 Chloramphenicol. The cultures were induced with 0.2 mM IPTG at an optical density (OD) 600 of 0.6–0.9, followed by an overnight incubation at 18 °C. Cells were harvested by centrifugation (5000 × g for 20 min at 4 °C) and resuspended in buffer containing 20 mM HEPES pH 7.5, 1 M NaCl, and 20 mM Imidazole. For cryo-EM studies, FnoCas12aKD2P was purified using a two-step method comprising Ni2+-NTA (nitrilotriacetic acid) and cation exchange chromatography. Cell lysis was achieved by sonication, and insoluble material was removed by centrifugation (30,000 × g for 45 min at 4 °C). The clarified supernatant was applied to a 5 mL Ni-NTA Superflow cartridge (Qiagen) equilibrated with wash buffer. After washing the resin with 20 column volumes of wash buffer, the bound protein was eluted from the Ni-NTA column using a continuous imidazole gradient, increasing to 100% buffer B (1X composition: 20 mM HEPES pH 7.5, 1 M NaCl, and 500 mM imidazole) over 15 column volumes. Eluted fractions were pooled and mixed with TEV protease for MBP-tag cleavage while dialyzing against cation exchange buffer A (1X composition: 20 mM HEPES pH 7.5, 150 mM KCl, 2 mM EDTA, and 1 mM DTT), overnight with slow stirring at 4 °C. Dialyzed protein was clarified by centrifugation and applied to a HiTrap SP HP cation-exchange column equilibrated in cation exchange buffer A. After washing with 10 column volumes of cation exchange buffer A, protein was eluted using a 0–100% cation exchange buffer B (1X composition: 20 mM HEPES pH 7.5, 1 M KCl, 2 mM EDTA, and 1 mM DTT) gradient over 20 column volumes, with FnoCas12a eluting at ~350 mM KCl. Fractions were assessed by 7.5% SDS-PAGE to confirm TEV cleavage (expected mass ~152 kDa). The purified protein was concentrated using the Thermo Scientific PierceTM protein concentrator PES, 30 kDa MWCO (2–6 mL) and the Cytiva Vivaspin 500 μL, 10 kDa MWCO centrifugal protein concentrators and stored on ice until complex preparation for cryo-EM. For variants used in biochemical assays, fractions eluted from cation exchange chromatography were pooled and further purified using size-exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR column; GE Healthcare) in 20 mM HEPES pH 7.5, 150 mM KCl, 2 mM EDTA and 1 mM TCEP. Peak fractions were concentrated using Amicon® Ultra-centrifugal filters (30 kDa MWCO, 15 mL), followed by final concentration to ~13 mg/mL with Cytiva Vivaspin filters (10 kDa MWCO, 500 µL). Proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. Aliquots were thawed on ice immediately prior to biochemical assays.

RNA and DNA preparation (for oligo sequences see Supplementary Table 22)
RNA was prepared by in vitro transcription and purified using urea polyacrylamide gel electrophoresis14,32,56. The template and T7 promoter DNA oligos (Supplementary Table 22) were ordered from Integrated DNA Technologies (IDT) and annealed in annealing buffer (10 mM HEPES, pH 8, 50 mM KCl) in a 1:1.5 molar ratio (2 μM: 3 μM) of template: T7 promoter strands. Approximately 400 ng of annealed template was used for in vitro transcription using 40 μg T7 RNA polymerase and NTPs (GTP: 9 mM, ATP: 8 mM, CTP: 8 mM, and UTP: 8 mM) in transcription buffer (1X composition: 40 mM Tris-HCl pH 8, 1 mM spermidine, 50 μg bovine serum albumin, 20 mM MgCl2, and 5 mM DTT)56. The transcription reaction was incubated at 37 °C for 4 h and then treated with 0.01 mg/mL DNase I to remove the DNA template. The crRNA product was precipitated through ethanol precipitation and further purified using a 12% polyacrylamide-8 M urea gel. The purified crRNA was suspended in annealing buffer and heated to 95 °C for 2 min, then slowly cooled to room temperature for correct folding and quantification. The folded crRNA was stored at 4 °C until use, up to a maximum of one week.

For cryo-EM, a 24-mer target strand (TS) and non-target strand (NTS) (Supplementary Fig. 8a) were ordered as ssDNA oligos from Sigma-Aldrich. The TS and NTS were annealed by mixing them in a 1:1 ratio (45 μM each) in annealing buffer, followed by heating at 95 °C for 5 min and allowing it to slowly cool to room temperature.

Cryo-EM grid preparation and data collection
Freshly purified 10 μM FnoCas12aKD2P protein and 12 μM crRNA were incubated at 37 °C for 30 min to form the ribonucleoprotein (RNP) complex in the complexing buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 1% glycerol, 1 mM TCEP, 2 mM EDTA). For formation of the ternary complex, 15 μM DNA duplex was added to the assembled RNP and incubated at 37 °C for another 30 min. The resulting ternary complex was loaded onto a Superdex 200 increase 10/300 GL column (GE Healthcare), which was pre-equilibrated with 1X complexing buffer at 4 °C, to separate complexes from excess nucleic acid. Peak fractions were analyzed on a 6% native polyacrylamide gel (40% (w/v) 29:1 acrylamide:bis-acrylamide) (Supplementary Fig. 1), and concentration was determined using absorbance at 280 nm as measured on a Nanodrop 8000 Spectrophotometer (Thermo Scientific), considering 1 Abs = 1 mg/mL. Peak fraction (~0.147 mg/mL, peak 2) containing the band for the ternary complex was vitrified on the same day of purification using a Leica EM GP2 plunger at 4 °C and 98% humidity. A 4 μL of sample was applied to graphene oxide (Sigma-Aldrich, 2 mg/mL)-coated Quantifoil R1.2/1.3 300 mesh Cu grid, which was glow discharged using PELCO easiGlowTM at 15 mA for 30 s (Preparation of graphene oxide grids is mentioned in Supplementary Method 1). After the sample application, the grid was immediately blotted for 2 s, followed by plunge freezing.

7232 micrographs were collected at the Stanford-SLAC Cryo-EM Center (S2C2), an NIH National Cryo-EM facility on TEM Gamma, a Titan Krios G3i (Thermo Fisher Scientific) cryogenic electron microscope operating at 300 keV and equipped with an X-FEG electron source, fringe-free optics, a BioQuantum (Gatan) energy filter and a K3 (Gatan) direct electron detector. Movies were recorded using a 100 μm objective aperture, an energy filter slit of 20 eV, and an electron dose of 1.25 e−/Å2 per frame at a magnification of 105,000× with a corresponding pixel size of 0.86 Å for a total exposure of 50 e−/Å2 (40 frames over 2.13 s, 0.053 s per frame) (Supplementary Table 1).

Single particle cryo-EM data processing and model building
The dataset was processed using the standard workflow on cryoSPARC v4.4.157. After patch motion correction and contrast transfer function (CTF) estimation, 5538 micrographs were selected for further processing. Particle picking, conducted with blob picker, identified 6,576,476 particles across all selected micrographs. Following particle inspection, 5,142,814 particles were extracted with a box size of 280 pixels (details in Supplementary Method 2).

We began 2D classification with 400 classes (details in Supplementary Method 2). After three rounds of iterative 2D classification, each with ~200 classes, we selected 399,609 particles for ab-initio reconstruction and homogeneous refinement. To assess conformational heterogeneity, 3D variability analysis was performed with a filter resolution of 4.5 Å. The variability observed in this step guided a subsequent 3D classification, resulting in six different classes (Supplementary Fig. 4, details in Supplementary Method 2), of which five, with up to 4 Å resolution, were used for structural analysis.

An initial model was built using PDB: 6GTG20 for each class. The models were first aligned with their respective maps on ChimeraX58, followed by manually rebuilding in Coot59 and refinement using Phenix.real_space_refine60,61. The REC1 and REC2 domains were individually isolated and fitted manually into maps, followed by rigid body fitting and real space refinement. The models were iteratively improved based on the validation metrics provided by MolProbity62 and Phenix refinement. The model building procedure for the FnoCas12aKD2P-S4a map, which exhibited stretched density for the REC2 domain and a different placement of REC2 compared to the corresponding position of this domain in PDB: 6GTE20, is described in section Supplementary Method 2.

In vitro DNA cleavage assays
Plasmid cleavage reactions (10 μL) were carried out in 1X plasmid reaction buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 2 mM TCEP, 10% glycerol, 5 mM MgCl₂). FnoCas12a and annealed crRNA were mixed to final concentrations of 25 nM and 50 nM, respectively, and pre-incubated at 37 °C for 10 min to form the ribonucleoprotein (RNP) complex. Reactions were initiated by the addition of 100 ng (~5 nM) substrate plasmid and incubation at 37 °C for 1–15 min, as indicated. Reactions were quenched by adding 10 μL of a 2X stop dye (1X composition: 50 mM EDTA, 1% SDS, 10% glycerol, 0.0125% orange G). Products were resolved on 1.5% agarose gels, post-stained with ethidium bromide (0.25 μg mL−1) and imaged using a Bio-Rad ChemiDoc system (Supplementary Fig. 17). Band intensities corresponding to nicked (IN), linear (IL), and supercoiled (ISC) species were quantified in ImageJ software63. The no-enzyme lane (consisting of DNA, buffer, and metal) of the same gel was used as a control, designated as ‘C’. Nicked, linear, and total cleavage percentages for each protein variant were calculated as:

Nicked (%) = [IN / (IN + IL + ISC)] * 100
Linear (%) = [IL / (IN + IL + ISC)] * 100
The fraction of total cleavage was defined as:
Total cleavage = Nicked (%) + Linear (%)

Standard deviation (SD) and standard error of the mean (SEM) were calculated as described previously13.

Three independent replications using proteins from two independent purifications were used in this work. Raw data (intensities of different DNA bands) and calculations are provided in the Source Data.

Computational modeling
We used structures of FnoCas12aWT states I2 (PDB: 6GTC20) and I4 (PDB: 6GTG20) and FnoCas12aKD2P states S2 and S4b for computational studies. The missing protein segments were added using Modeller 10.664, protonation states were assigned by PROPKA65,66, and systems were solvated, neutralized, and prepared, maintaining consistency with experimental conditions. Each system underwent 1 μs NVT simulation. The resulting trajectories, totaling 4 μs after including all the systems, were concatenated and analyzed using the Principal Component Analysis (PCA). For the cross-correlation analysis, the first 100 ns of the initial NVT production trajectory was excluded and only the last 900 ns of each trajectory were used. Both PCA and cross-correlation analyses were performed using CPPTRAJ67. Simulations were performed using the AMBER2068 employing AMBER/ff14SB, OL15, ff99bsc0+χOL3, and TIP3P parameters for protein, DNA, RNA, and water, respectively69–71. Protonation states were assigned using PROPKA, and systems were solvated in TIP3P water using a nearly cubic simulation box with at least 12 Å of solvent beyond the protein/crRNA/DNA complex in all directions, neutralized with Na+ counterions, and prepared without divalent metals to match experimental conditions. Each system was heated to 300 K and equilibrated through sequential NVT and NPT stages (1 ns NVT heating followed by NPT equilibration at 1 bar, continued until the system density reached ~1.01 g/cm3) before initiating the 1 μs NVT production run. Additional computational details are provided in Supplementary Method 3.

Bioinformatics
HMM (Hidden Markov Model) profile for the BH–RuvC-H1 region of FnoCas12a (residues 953-1000; Uniprot ID A0Q7Q2) was built using HMMER40 based on the sequence alignment using Clustal Omega72 for 313 Cas12a annotated sequences retrieved from NCBI (National Centre for Bioinformatics Information). The profile was used to search for similar sequences using hmmsearch (a tool in HMMER) with a default e-value of 0.01. Structural similarity search for the BH–RuvC domain (motifs I, II, and III; PDB: 5NFV23) was performed using DALI server41. The top hits belong to Cas12a orthologs with Z-scores ranging from 24.9 to 13.9, while hits belonging to other Cas12 subtypes, bacterial and viral Resolvases, Recombinase, and TnpB have Z-scores ranging from 13.5 to 8.0 indicating there is probable structural homology40. To assess the structural similarities of Cas9, the BH and RuvC domain of eight Cas9 structures belonging to subtypes II-A, II-B, and II-C were added to the Cas12 structures that were identified by DALI search. Structures that belong to similar bacterial species were removed to avoid redundancy in the structure comparison. This was followed by an “all-against-all structure comparison” for the selected Cas12 and Cas9 structures covering the BH and RuvC domain (sequences covering this region are available in Source Data). The output from DALI was visualized as a dendrogram in a rectangular mode (unrooted) using an online tool iTOL73 (Interactive Tree of Life). The PDB codes and the corresponding structures used for structural comparison are indicated in Fig. 5, Supplementary Fig. 23, and Supplementary Table 23.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript provides a detailed structural analysis of the bridge helix (BH) in Cas12a activation, offering insights into its role in allosteric regulation. The cryoEM data are well-analyzed, and the findings contribute meaningfully to the understanding of Cas12a’s conformational transitions. The study is of interest to researchers in CRISPR biology and genome editing. While the manuscript is generally well-prepared, some points require further clarification and discussion to strengthen the conclusions.\n\nMajor Comments\n1. The study suggests that BH’s loop-to-helical transition directly influences RuvC activation. However, it remains unclear whether this is a cause or a consequence of R-loop formation. Additional discussion, or reference to supporting biochemical evidence, would help clarify this point.\n2. The manuscript suggests that the impaired BH prevents trans-cleavage, but no direct biochemical validation is provided. If prior studies have addressed this, a more explicit reference would be beneficial. Otherwise, acknowledging this limitation would improve clarity.\n3. The role of BH in Cas12a is emphasized, but how this differs from Cas9 remains unclear. Since Cas9 does not require a major RuvC conformational shift, a brief discussion on this evolutionary divergence would add depth to the paper.\n4. Cas12a’s ability to recognize mismatches is only briefly mentioned. A more detailed discussion would be useful, especially given its implications for high-fidelity genome editing.\n\nMinor Comments\n1. In the abstract, the authors state, “Overall, our study enables the development of high-fidelity Cas12a and Cas9 variants by BH modifications.” However, the study does not provide direct data on BH regulation in Cas9, making this claim difficult to support. Clarification or a more cautious phrasing would be advisable.\n2. Suggest the authors to enhance the figures clarity further. In many figures, the information is not sufficient. For example, Figure 2, what color indicates what component? What the state it is for Panel D to G. I noticed the authors mentioned it in the figure legend, however, this will affect the reading experience for the figure. If the author can label the component in the figure according to the color, that would be great. Many figures have this similar issue.\n3. In the Figure 2, the display of W971 is unclear. Because this is very important point, A clear cryoEM density of the residues to show the interaction distances would be helpful.", "reviewer_2": "Ganguly et al. describes the potential importance of the bridge helix (BH) domain of the Cas12a from Francisella novicida (FnoCas12a) in mediating conformational state transitions throughout initial R-loop formation, implicating it as an allosteric activator of REC domain movement and in achievement of the enzyme’s final catalysis-primed conformation. The authors present a series of five (very nice, often high resolution) conformationally unique structures of a mutated FnoCas12aKD2P model system. These demonstrate the domain movements associated with the extension of the R-loop as it forms and how the CRISPR effector undergoes a large scale ‘opening’ in order to accommodate this extending heteroduplex. The structures are of varying quality, the authors interpreted the density reasonably.\n\nSuitability for Nature Communications\n• Given the structural focus of this manuscript, I would recommend publication in a more suitably specialized journal (i.e. Structure). This manuscript is purely focused on structural biology and, as such, would be a better fit for journals more so catered to this focus. More biochemical analysis would be required for a broader audience.\n\nMajor Comments\n• The authors rightly bolster their results through comparison to similar structures in Stella et al., 2018, and Strohkendl et al., 2024, which describe conceptually similar findings focused on FnoCas12a and AsCas12a, respectively. However, it is unclear in this manuscript what is the new intellectual contribution aside from the role of this mutated bridge helix. The manuscript would benefit from a clearer presentation of what is new. .\no Further, the two referenced manuscripts described above resolve structures of WT enzymes, whereas this manuscript focuses on a Cas12 variant with a modified bridge helix domain thought to slow kinetics of conformational transitions. While this mutant system may be descriptive of WT mechanisms, the lack of biochemical confirmation of their findings makes it challenging to distinguish if these lessons learned are broadly applicable or simply idiosyncrasies of an impaired system.\no While the authors have made efforts to biochemically characterize this specific mutant of FnoCas12a (Priya et al. 2021), and indeed some results from this previous manuscript strengthen claims made here, their previous work was largely focused on catalysis with little biochemical evidence to directly support the claims made in this manuscript which implicate the bridge helix in pre-catalysis conformational changes. More biochemistry is required here.\n• The figures in this manuscript are simplified to the point where it makes their interpretation challenging. Figures containing protein structures are under-labeled and stripped of context, requiring frequent reference to the manuscript, figure legend, and occasionally the provided structures, to properly understand. Increased labeling (as seen in figure 5B) and structural context callouts (as seen in figure 2A) would go a long way in increasing the clarity of the images to the reader.\n\nMinor Comments\n• The authors note on page 17 that their enzyme FnoCas12aKD2P is unable to perform trans-cleavage. Priya et al. 2021 demonstrates that while trans-cleavage is dramatically inhibited in FnoCas12aKD2P it does still occur if incubated for long enough – at least with M13 ssDNA.\n• While the bioinformatics was well done, I thought that, in line with my comments above, the associated figures highlighting structural similarities were unclear (though these were much better than previous figures). Further, the commentary on the bioinformatics portion of this manuscript was surface level and somewhat vague with little discussion beyond how they are structurally (and thus potentially mechanistically) similar. We suggest that the implications of this bioinformatic analysis be explored in the discussion section. For example, how frequently do these domain insertions occur across similar systems?\n• We found typos/grammatical errors in the manuscript, please proofread carefully.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank all the reviewers for their positive feedback about the significance of the work and their critical comments that have greatly helped us improve the quality of our manuscript and distinguish the contributions of this manuscript over the previously existing literature. A point-by-point response to the reviewer comments is below in blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript provides a detailed structural analysis of the bridge helix (BH) in Cas12a activation, offering insights into its role in allosteric regulation. The cryoEM data are well-analyzed, and the findings contribute meaningfully to the understanding of Cas12a’s conformational transitions. The study is of interest to researchers in CRISPR biology and genome editing. While the manuscript is generally well-prepared, some points require further clarification and discussion to strengthen the conclusions.\nWe thank the reviewer for the positive feedback about the significance of the work and the quality of cryo-EM data analysis and its contribution to understanding the role of BH in Cas12a’s activation. Points which needed further clarification are addressed in the revision as described below.\nMajor Comments\n1. The study suggests that BH’s loop-to-helical transition directly influences RuvC activation. However, it remains unclear whether this is a cause or a consequence of R-loop formation. Additional discussion, or reference to supporting biochemical evidence, would help clarify this point.\nTo clarify whether BH’s loop-to-helical transition is a cause or a consequence of R-loop formation and its influence on RuvC activation, we have conducted additional biochemical, structural, and computational analyzes. The results now clearly establish that BH’s loop-to-helical transition is a cause since without this transition, R-loop formation is inhibited mid-way through, and the formation of the helical structure of the hybrid is impaired. We also show that, with a variant able to undergo the loop-to-helical transition, BH–RuvC-lid interactions are only secondary as the variants maintain efficient on-target DNA cleavage without the BH–RuvC-lid interactions.\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all my previous comments. The revised manuscript is clearly improved in both presentation and data interpretation. The additional experiments and clarifications have strengthened the conclusions, and the responses were detailed and appropriate.\nI have no further concerns.", "reviewer_4": "We thank all the reviewers for their positive feedback about the significance of the work and their critical comments that have greatly helped us improve the quality of our manuscript and distinguish the contributions of this manuscript over the previously existing literature. A point-by-point response to the reviewer comments is below in blue. REVIEWER COMMENTS Reviewer #1 (Remarks to the Author): This manuscript provides a detailed structural analysis of the bridge helix (BH) in Cas12a activation, offering insights into its role in allosteric regulation. The cryoEM data are well-analyzed, and the findings contribute meaningfully to the understanding of Cas12a’s conformational transitions. The study is of interest to researchers in CRISPR biology and genome editing. While the manuscript is generally well- prepared, some points require further clarification and discussion to strengthen the conclusions. We thank the reviewer for the positive feedback about the significance of the work and the quality of cryo-EM data analysis and its contribution to understanding the role of BH in Cas12a’s activation. Points which needed further clarification are addressed in the revision as described below. Major Comments 1. The study suggests that BH’s loop-to-helical transition directly influences RuvC activation. However, it remains unclear whether this is a cause or a consequence of R-loop formation. Additional discussion, or reference to supporting biochemical evidence, would help clarify this point. To clarify whether BH’s loop-to-helical transition is a cause or a consequence of R-loop formation and its influence on RuvC activation, we have conducted additional biochemical, structural, and computational analyzes. The results now clearly establish that BH’s loop-to-helical transition is a cause since without this transition, R-loop formation is inhibited mid-way through, and the formation of the helical structure of the hybrid is impaired. We also show that, with a variant able to undergo the loop-to- helical transition, BH–RuvC-lid interactions are only secondary as the variants maintain efficient on- target DNA cleavage without the BH–RuvC-lid interactions. 1 Biochemical Analysis: To probe the allosteric mechanism, residues that mediate BH-lid interactions to convert the lid from a loop to a helical form at different stages of the activation process, thereby mediating the exposure of RuvC active site, were identified based on wild-type (FnoCas12aWT) and BH-variant (FnoCas12aKD2P) structures. Residues R964 of the BH and E1020 and K1021 of the RuvC-lid were substituted with alanine, along with a double variant E1020A/K1021A in both FnoCas12aWT and FnoCas12aKD2P backgrounds. In vitro plasmid DNA cleavage assays using both matched (MCH) and mismatched (MM) DNA substrates (MM2, PAM proximal mismatch and MM13, mid R-loop mismatch) were performed to assess the effects of the alanine substitutions. In the FnoCas12aWT background, single substitutions do not show prominent impairments, except for MM13-R964A combination, consistent with the stabilizing effect that the BH offers for the allosteric interactions. On the other hand, in the FnoCas12aKD2P background, where BH stability is impaired by the proline substitutions, the substitutions at R964 of the BH and the double variant (E1020/K1021) of the RuvC-lid, impacted both TS and NTS cleavages. In comparison, the only significant impact seen in FnoCas12aWT is the accumulation of nicked intermediate in R964A-MM13 combination (Figures 3e-f, Supplementary Figs.17,18, and Supplementary Table 11). These data clearly show that DNA cleavage is an additive process with combinations from effects from the BH’s loop-to-helical transition, opening of the RuvC active site by the helical transition of the lid, and the strength of complementary pairing between the RNA and DNA. Of these three, BH’s transition has the most significant contribution since in the wild-type backbone, all substitutions show minimal effect, except in R964-MM13, which indicates that the second contributor is the strength of complementary base pairing that is also dictated by the position of the mismatch between the RNA and DNA. The new results are presented in pages 12-15, with a summary in pages 14 (lines 23-26, and 15 (lines 1-10) which is copied below. “Our findings show that contacts between the BH and RuvC-lid (R964-E1020, E961-K1021) are only secondary to the BH’s helical transition for orchestrating the conformational transitions required for double-strand cleavage and mismatch sensitivity. The wild-type protein with an intact conformational change in the BH can linearize (i.e., both NTS and TS cleavages) despite disrupting the BH–RuvC-lid interactions, with the only significant reduction being in the second strand cleavage for MM13 substrate (Supplementary Fig. 17 and Supplementary Table 11). On the contrary, when BH’s sturdiness is restricted, as in the FnoCas12aKD2P variant, the enzyme becomes highly dependent on these interactions to provide RuvC active site access to ssDNA. In the FnoCas12aKD2P background, while R964A and E1020A/K1021A substitutions impair both strand cleavages, single substitutions (E1020A or K1021A) impede second-strand cleavage (Supplementary Fig. 18a and Supplementary Table 11). These 2 results demonstrate that the BH’s loop-to-helical transition is an allosteric switch coupling R-loop propagation to catalysis and point to the importance of the helicity of the BH in assisting the passage of TS DNA into the RuvC active site pocket. BH, RuvC-H1, and RuvC-lid are potential engineering sites to reduce mismatch tolerance in Cas12a.” Structural Analysis: Structural analysis showed that R964 and R968 interact with nucleotides dG(-7), dT(-8), and U10 of crRNA/DNA in state I4 of FnoCas12aWT (Supplementary Fig.15), while the corresponding interaction is absent in FnoCas12aKD2P (a maximum of 11 base pairs is visible in State 4a). While position 11 of crRNA and TS DNA move closer to the BH in FnoCas12aWT, this interaction stays at around 20 Å FnoCas12aKD2P. Previous studies have shown that 16 nucleotides of base pairing of crRNA and DNA is a checkpoint for Cas12a activation (Stella et al., 2018 and Strohkendl et al., 2024). Thus, it appears that BH’s transition is essential for R-loop propagation, making BH’s transition the cause for R-loop propagation, especially past 10-nucleotides of base pairing. The bending of BH towards R-loop and latching into REC2 domain in parallel with the growing RNA-DNA hybrid appears to give the RNA-DNA hybrid a double-helical structure, implicating the importance of BH in R-loop propagation and stability. This stable configuration of the BH and the R-loop seems to facilitate a concomitant opening of the RuvC active pocket through RuvC-lid remodeling. These points have been added to the Discussion section (page 19 (lines 14-26), which is copied below). “In addition, the elongation of the BH due to the addition of the new helical turns and its bending towards the RNA-DNA hybrid appears to be critical in providing an anchorage for R-loop and to complete the RNA-DNA hybrid formation. In the FnoCas12aWT structures, states I2 and I320 have BH in a loop state prior to its the helical conversion. In these structures, BH is at a distance of 17 Å from the RNA-DNA hybrid with a total of 8-nucleotides of base pairing between the crRNA-guide and TS DNA. In comparison, in state I4, we see a fully formed and bent BH that is closer to the RNA-DNA hybrid (6 Å). In FnoCas12aKD2P, which is not capable of undergoing the loop-to-helical transition, we only see up to a maximum of 11-nucleotides of base pairing between the crRNA-guide and TS DNA, with a distance of ~20 Å from the BH. Interestingly, we also see stacking and distortion of bases towards the end of the R- loop, implicating that the strands are not aligned well with an impaired BH, hinting at the essentiality of the stability provided by the BH in these intermediary stages to drive R-loop propagation. The slowing down of the structural transitions is supported by the lower reaction kinetics of FnoCas12aKD2P when compared to FnoCas12aWT14..” 3 Computational Studies: ⁠Our MD simulations indicate that the BH’s loop-to-helical transition actively shapes the conformational landscape of Cas12a by exerting allosteric influence over the RuvC-lid, crRNA, and DNA. Cross- correlation analyses show a shift from anti-correlated to positively correlated interactions across states, such as S2 to S4b to I4 (especially for nucleotides at position 6 and 8 of crRNA and -8 to -4 of DNA). This reorganization coincides only with the helical transition of the BH, which in turn facilitates the emergence of strong BH–crRNA, BH–DNA, and BH–RuvC-lid couplings in the pre-catalytic state. Overall, these data show that BH’s helical transition is an activating trigger regulating R-loop propagation and opening of RuvC active site pocket (Points have been added in the Results section, page 16 (lines 8-24), which is copied below, and Supplementary Methods 3). “To delineate potential allosteric communication between BH and nucleic acid, we performed correlation analyses of the residues E961, R964, R968, K981, E1020, K1021, and N1022 of the BH, RuvC-H1, and RuvC-lid, as well as nucleotides in crRNA and DNA (Fig. 4c-e). The complete correlation analysis is available in Supplementary Methods 3. Results reveal that BH–RuvC-H1 and RuvC-lid conformations exhibit strongly correlated motions with those of the crRNA and DNA, and that the motions are different between the different states. FnoCas12aKD2P S2 has fewer correlated motions (i.e., components move in the same direction) between the BH–RuvC-H1 and RuvC-lid, while there are several anti-correlated motions (i.e., components move in the opposite direction) between BH–RuvC-H1 and the RNA and DNA (Fig. 4c). In the intermediate state S4b, however, this correlation pattern shifts; there is an increase in the correlated motions between BH–RuvC-H1 and RuvC-lid and a few new motions with the crRNA, while there still is a substantial number of anti-correlated motions with crRNA and DNA (Fig. 4d). In I420 of FnoCas12aWT, we see only correlated motions between BH–RuvC-H1, RuvC-lid, crRNA, and DNA (Fig. 4e). Interestingly, some of the interactions with crRNA and DNA include nucleotides near the middle of the R-loop (~8) providing mechanistic clues on why mismatches between the crRNA and DNA in the R-loop away from the PAM are particularly sensitive in FnoCas12a. This reorganization of the correlation network in different states of the protein points to the pivotal role that the BH–RuvC-H1 transitions play in organizing the cleavage-competent form of Cas12a.” 2. The manuscript suggests that the impaired BH prevents trans-cleavage, but no direct biochemical validation is provided. If prior studies have addressed this, a more explicit reference would be beneficial. Otherwise, acknowledging this limitation would improve clarity. 4 We thank the reviewer for this helpful comment. While the current manuscript focuses on providing structural insights into the cis DNA cleavage mechanism of FnoCas12aKD2P, previous biochemical evidence from our group (Parameshwaran et al., 2021) reported that this variant has significantly reduced trans-cleavage efficiency compared to FnoCas12aWT). We now cite and discuss this study more explicitly in the discussion section to show that the BH transition and the corresponding allosteric activation of RNA/DNA/RuvC-lid are important for trans DNA cleavage as well. We have carefully worded the text to make it clearer to a reader that trans cleavage assay was done in our prior work and that the structural data provides the mechanistic framework that explains the previously observed defect (Points have been added in the Discussion section, page 21, lines 1-9, which is copied below). “In our previous work, we have also demonstrated that FnoCas12aKD2P has a lower trans-cleavage efficiency (0% substrate left for FnoCas12aWT at 5 minutes, compared to 40% remaining in FnoCas12aKD2P after two hours)14. Unlike FnoCas12aWT, which rapidly degraded substrates, FnoCas12aKD2P accumulated linear intermediates and failed to sustain a robust progressive trans- cleavage14. Additionally, FnoCas12aKD2P was unable to perform trans-cleavage when activated by a dsDNA14. The trans-cleavage occurs through keeping the RuvC domain “open” for non-specific ssDNA to access the active site24,53. These observations highlight that impairment of the BH’s helical transition likely blocks the prolonged activated form of the RuvC pocket, preventing trans-cleavage in FnoCas12aKD2P.” 3. The role of BH in Cas12a is emphasized, but how this differs from Cas9 remains unclear. Since Cas9 does not require a major RuvC conformational shift, a brief discussion on this evolutionary divergence would add depth to the paper. We thank the reviewer for this comment. It prompted us to include Cas9 proteins in our structural analyses. Further analyses by including the available structures of Cas9 subtypes (II-A, II-B and II-C) in using the pairwise 3D structural comparison of the BH-RuvC domain of FnoCas12a and various Cas12 subtypes (Fig. 5 and Supplementary Fig. 22) suggest that the Cas9 BH do not align completely with that of FnoCas12a, whereas a helix in the RuvC motif-II is structurally positioned in a similar arrangement as the RuvC-H1 of Cas12a. As the BH and the RuvC motif-II are placed farther away from each other due to the insertion of the REC lobe in between in Cas9, it may not be feasible to translate or couple the conformational changes in the BH to the RuvC motif-II’s helix or vice versa, similar to the coordinated transitions that we observe in Cas12a’s BH and H1 (Supplementary Fig. 23). In Cas12a, the BH and the RuvC-H1 of the RuvC motif-II are located close to each other both in the primary 5 sequence and the tertiary structure, facilitating such coordinated transitions. It should be noted that there is a loop to helical transition seen in the long BH of Cas9, which we have shown in our previous studies to possess an allosteric effect in DNA cleavage mechanism and RNA-DNA mismatch sensitivity (Babu et al., 2019, Babu et al., 2021 and Wu et al., 2025). Thus, while the BH’s helical transition and its allosteric effect on DNA cleavage seems to be a conserved mechanism in Cas9 and Cas12a, the structural features contributing to this allostery seem to be evolutionarily divergent. We have included these results and discussions in our revised manuscript [e.g., Results section, page 18 (lines 3-19), which is copied below]. “Structural overlays of the BH and RuvC domain (motifs I, II, and III) of Cas9 (subtypes II-A, II-B, and II- C) with those of FnoCas12a show that the BH and a helix of the RuvC motif-II of Cas9 have a similar structural arrangement to that in Cas12a. While helix in the RuvC motif-II of Cas9 and Cas12a RuvC- H1 shows a better structural overlay, the BH of Cas9 is prominently longer and does not align with that of FnoCas12a (Fig. 5b, Supplementary Fig. 22-23). Another significant difference between Cas9 and Cas12 is the insertion of the REC lobe between BH and RuvC motif-II, compared to their direct connection in Cas12 families (Supplementary Fig. 23). This long separation can potentially disrupt coordinated conformational changes between BH and RuvC-H1 that are present in Cas12s. Within the Cas9, there is a loop-to-helical transition of the BH in response to RNA binding that has been demonstrated to impact DNA cleavage efficiency and DNA mismatch sensitivity6,11,13,32,33,52. These observations show that while the fundamental mechanism of loop-to-helical transition of the BH is conserved in Cas12a and Cas9, the mechanisms for coordinated conformational changes differ between them. While the RuvC-H1 of RuvC motif-II is co-opted by Cas12 families to undergo coupled transformations with the BH to orchestrate conformational changes needed for assembling the pre- catalytic state, such coupling is absent in Cas9 (Supplementary Fig. 23c). Thus, nature seems to have optimized Cas12a and Cas9 molecular mechanisms differently to attain the same goal of RNA-directed, sequence-specific, DNA cleavage.” 4. Cas12a’s ability to recognize mismatches is only briefly mentioned. A more detailed discussion would be useful, especially given its implications for high-fidelity genome editing. We thank the reviewer for raising this important point. To address this, we have expanded the discussion to highlight prior biochemical evidence that demonstrated FnoCas12aKD2P’s RNA-DNA mismatch sensitivity that was reported in our previous work (Parameshwaran et al., 2021). Additionally, we 6 included two selected DNA mismatches (MM2 and MM13, representing PAM-proximal and PAM-middle DNA mismatch) to characterize the on-target vs. off-target cleavage effects of the new variants that were created during the revision of this manuscript. These data show that combining BH and RuvC-lid amino acid substitutions exacerbate mismatch sensitivity and provides both structural and biochemical support for a BH-dependent checkpoint that enforces the DNA cleavage fidelity of Cas12a. [Discussion section, page 20 (lines 5-26), which is copied below]. “In another role, the loop-to-helical transition of RuvC’s lid has been shown to play an important role in bringing the TS strand into the active site pocket for DNA cleavage21,27. The AsCas12a structures probing the TS strand entry mechanisms (PDB: 8SFP, 8SFQ and 8SFR21) also support these since the BH is extended towards the RNA-DNA hybrid, establishing interactions with the RuvC-lid helix, which leads to the exposure of the active site pocket in these structures. While we do not have structural features to support the TS entry mechanism due to the presence of partial DNA strands, BH’s helical transition appears to be critical in the passage and cleavage of the TS. This is supported by our previous work and biochemical assays reported in the present work. We observed a 6-fold reduction in TS compared to only a 3-fold reduction in NTS cleavage by FnoCas12aKD2P compared to that by FnoCas12aWT 14. Additionally, FnoCas12aKD2P discriminates the RNA-DNA mismatches by preventing TS cleavage since it was able to nick substrates with mismatches but not linearize them14. The efficiency to cleave mismatches is lower for all 20 nucleotides along the RNA-DNA hybrid, with mismatches at positions 12-17 of the target DNA producing higher accumulation of the nicked intermediate (61-77%) compared to that in FnoCas12aWT (3-19%)14. In this study, we observe that variants with disrupted BH–RuvC-lid interactions (R964A, E1020A/K1021A) in the FnoCas12aKD2P background exhibit pronounced reductions in cleavage of both TS and NTS, while the significant effect observed in FnoCas12aWT is the reduction in linearization and accumulation of nicked intermediate for MM13 substrate. Together, these results indicate that the BH’s loop-to-helical transition is the main driving force in R-loop propagation and the opening of the RuvC-lid in response to the total length of the RNA-DNA hybrid. Further studies are needed to find the mismatch-sensing mechanism of FnoCas12aKD2P, especially to understand why positions 12-18 of the RNA-DNA paired region is more sensitive compared to the rest of the R-loop positions.” Minor Comments 1. In the abstract, the authors state, “Overall, our study enables the development of high-fidelity Cas12a and Cas9 variants by BH modifications.” However, the study does not provide direct data on BH 7 regulation in Cas9, making this claim difficult to support. Clarification or a more cautious phrasing would be advisable. We thank the reviewer for pointing this out. To address this concern, we have modified the sentence in the abstract (page 2, lines 13-15) as follows: “Pairwise 3D structural comparison of the BH and RuvC of Cas12a and Cas9 provides insight into the diversity of BH-mediated DNA cleavage in these mechanistically similar enzymes”. 2. Suggest the authors to enhance the figures clarity further. In many figures, the information is not sufficient. For example, Figure 2, what color indicates what component? What the state it is for Panel D to G. I noticed the authors mentioned it in the figure legend, however, this will affect the reading experience for the figure. If the author can label the component in the figure according to the color, that would be great. Many figures have this similar issue. We thank the reviewer for this helpful suggestion. We agree that increasing figure clarity will improve readability of the manuscript. In the revised version, we have updated the figures by directly labeling components within the panels so that readers can readily identify which colors correspond to which domains or states without needing to refer to the legend. In Figure 2, we now provide explicit in-panel labels for the BH, RuvC-H1, crRNA, and DNA across panels a-c, with consistent color-coding across all figures. We have also ensured that similar improvements are made to other multi-panel figures where components or states were previously shown in different colors. These revisions should substantially enhance the clarity and self-contained interpretability of the figures. 3. In the Figure 2, the display of W971 is unclear. Because this is very important point, A clear cryoEM density of the residues to show the interaction distances would be helpful. We thank the reviewer for this important suggestion. In the revised manuscript, we have improved the visualization of W971 by providing a clearer cryo-EM density map and explicitly displaying the interaction distances. To avoid overcrowding the main figure, these panels have been moved to the supplementary information (Supplementary Figs. 13a–d). This updated representation should allow readers to more clearly evaluate the density corresponding to W971 and assess its positioning relative to neighboring residues. We have shown states S1 and S4b of FnoCas12aKD2P in the figures since the density for the sidechain of residues W971 and Y579 are not visible in S2 and S4a and, in state S3, densities for both 8 BH and REC2 domain are absent. These are described in the main text [Results section, pages 11, (lines 22-26), and 12 (lines 1-3) which is copied below] is as follows: “Another important conformational change towards activation of Cas12a is the insertion of the conserved W971 (residue immediately following the proline substitutions of FnoCas12aKD2P at 969 and 970) into the REC2 hydrophobic pocket (composed of amino acids Y579, K527, K524, R583 of FnoCas12aWT 12,16,22) to enable REC2 docking after hybrid formation. In FnoCas12aKD2P structures, we see insertion of W971 into the REC2 hydrophobic pocket in S1 and S4b (S2 and S4a do not show density in this area to determine sidechain position). However, the interacting distance of W971 with Y579 of the REC2 domain, which is at ~4 Å in FnoCas12aWT, remains farther in S1 and S4b (6 Å and 10 Å, respectively) (Supplementary Fig. 13).” 9 Supplementary Figure 13: BH transitions are important for REC2 domain docking. (a-d), The conserved residue W971, immediately following the K969 and D970 (969 and 970 were substituted with proline in FnoCas12aKD2P), docks into the REC2 hydrophobic pocket (composed of amino acids Y579, K527, K524, and R583) and interacts closely with Y579. Close-up view of the impaired BH of FnoCas12aKD2P impacting this interaction in a, S1 and b, S4b where W971 and Y579 are at a distance of 6 Å and 10 Å, respectively. Zoomed in view of electron density from the cryo-EM maps to show the sidechain density for residues W971 and Y579 in c, S1 and d, S4b. Close interaction between W971 10 and Y579 is observed in both a, binary (PDB: 5NG610) and b, full R-loop states (PDB: 6GTG4) in FnoCas12aWT. Two REC2 helices (V523-H538 and F565-T587, dodger blue), BH (pink), and RuvC-H1 (purple) are shown in this figure. 11 Reviewer #2 (Remarks to the Author): Ganguly et al. describes the potential importance of the bridge helix (BH) domain of the Cas12a from Francisella novicida (FnoCas12a) in mediating conformational state transitions throughout initial R-loop formation, implicating it as an allosteric activator of REC domain movement and in achievement of the enzyme’s final catalysis-primed conformation. The authors present a series of five (very nice, often high resolution) conformationally unique structures of a mutated FnoCas12aKD2P model system. These demonstrate the domain movements associated with the extension of the R-loop as it forms and how the CRISPR effector undergoes a large scale ‘opening’ in order to accommodate this extending heteroduplex. The structures are of varying quality, the authors interpreted the density reasonably. We thank the reviewer for the positive feedback on the quality of the structures, the interpretation of the density, and the explanation about domain movements with respect to R-loop propagation. Suitability for Nature Communications 1. Given the structural focus of this manuscript, I would recommend publication in a more suitably specialized journal (i.e. Structure). This manuscript is purely focused on structural biology and, as such, would be a better fit for journals more so catered to this focus. More biochemical analysis would be required for a broader audience. We thank the reviewer for their critical comments. To improve the suitability of our manuscript to Nature Communications, in the revision, we have added detailed biochemical and computational analyses as well as deepened our bioinformatics analysis to include a comparison between the BHs of Cas12 and Cas9 families. The results further strengthen the structural insights from the cryo-EM data and provide experimental evidence for RuvC–BH and BH–RNA/DNA interactions. Finally, the computational results establish a cooperativity between all the three components (BH, RuvC-lid, RNA-DNA hybrid) providing the first direct evidence of BH’s allosteric mechanism in organizing a DNA cleavage competent state of Cas12a. The main changes are: Based on our structural analyses, we created single and double variants of FnoCas12aWT and FnoCas12aKD2P and performed plasmid cleavage assays with BH–RuvC-lid interface variants (R964A, E1020A, K1021A, E1020A/K1021A) in both FnoCas12aWT and FnoCas12aKD2P backgrounds. We tested the impact of these substitutions on mismatch discrimination in addition to on-target DNA cleavage to 12 support our structural interpretations. Details are present in Results section, page 12, lines 12 to page 15 line 10) with a summary in pages 14 (lines 23-26) and 15 (lines 1-10), which has been shown in page 2 of the rebuttal to address Major Comment 1 of Reviewer #1, biochemical analysis. In support of our biochemical and structural analyses, our molecular dynamics simulations show that the loop to helical transition of the BH reshapes the conformational landscape of FnoCas12a by altering correlation networks between the BH, RuvC-lid, crRNA, and DNA. This highlights BH’s loop-to helical transition as the allosteric trigger needed to propagate R-loop beyond 15 nucleotides of pairing and the subsequent activation of Cas12a for DNA cleavage (i.e., loop to helical transition of RuvC-lid exposing the active site for DNA entry). Points have been added in the Results section, page 16 (lines 7-24), and Supplementary Methods 3, which has been shown in page 4 of the rebuttal to address Major Comment 1 of Reviewer #1, computational studies). Our bioinformatics analysis revealed similar structural organization of BH and the first helix of RuvC motif-II as in Cas12a across all Cas12 subtypes. Interestingly, our new analyses including Cas9 show that while there is a helix in the RuvC motif-II of Cas9 subtypes that is positioned in a similar arrangement as that of the RuvC-H1 of Cas12a’s RuvC-motif-II, the coordinated conformational changes between RuvC helix and BH is absent in apo, binary, and ternary structures of Cas9. The insertion of the large REC lobe between the BH and RuvC motif-II’s helix may deter such allostery between the BH and RuvC in Cas9. We point to our previous work on Cas9 BH’s conformational change that involves a loop-to-helical transition within the BH domain, alluding to the mechanistic similarities and structural differences of these divergent Cas proteins. Details are present in Results section, page17, line 3 to page18, line 19, a summary of which has been shown in pages 5-6 of the rebuttal to address Major Comment 3 of Reviewer #1. We believe that incorporating these complementary approaches will make the study relevant to a broad readership interested not only in Cas12a structure but also in its biochemical mechanism, DNA cleavage fidelity, and potential applications in genome editing. Major Comments 2. The authors rightly bolster their results through comparison to similar structures in Stella et al., 2018, and Strohkendl et al., 2024, which describe conceptually similar findings focused on FnoCas12a and AsCas12a, respectively. However, it is unclear in this manuscript what is the new intellectual contribution 13 aside from the role of this mutated bridge helix. The manuscript would benefit from a clearer presentation of what is new. We thank the reviewer for this critical comment since that has helped us to differentiate our contributions and place them rightly among those from the previous literature. Here is a list of the main findings from our work and how it relates to previous literature. • The previous studies reported the structures of the wild-type (WT) Cas12a orthologs in several states. Our structures capture several conformational intermediates that are different than those that were captured in the previous work. These structures demonstrate how the inability of the BH’s loop-to-helical transition traps the protein in conformational intermediates compared to what was seen in WT under the normal conformational cascade. These structures directly connect the BH’s conformational change as a necessity for R-loop formation past the checkpoint (around 16 nucleotides), and how it is intimately related to RuvC-lid opening. The results also show that the structures of the BH-impaired variant are able to capture the earlier states (I1 to I3 of FnoCas12aWT) in the conformational cascade, but not the later ones, providing direct evidence that BH is an allosteric regulator for the conformational coordination towards the later stages of the activation process. Thus, while WT structures offer conformational changes needed for Cas12a activation, the importance of BH’s conformational change and its allosteric control is not discernible only by comparing the WT structures. • We also see slight differences in the BH’s conformational changes among the different orthologs. In FnoCas12a, the BH undergoes a loop-to-helical transition in response to R-loop propagation followed by the bending of the BH to latch onto the growing R-loop. However, in AsCas12a and LbCas12a, the BH remains elongated in the binary and the initial conformational states and bends toward the R-loop post 16 nucleotides of base pairing that is a conformational checkpoint in the activation process (Supplementary Fig. 24). Thus, the BH’s loop-to-helical transition in FnoCas12a appears to serve as an additional structural sensor for RNA-DNA hybrid propagation, melting of RuvC’s H1, and opening of RuvC-lid that are all needed for catalytic activation. • The functionally important W971 that inserts into the REC2 domain in the later stages of catalytic activation is the residue right adjacent to the BH, located in-between the BH and RuvC H1. Our structures show that an impaired loop-to-helical conformational change of the BH prevents 14 insertion of W971 to REC2’s hydrophobic pocket that implicates that proper REC2 docking is facilitated by BH’s conformational change. • We validated our structural findings through biochemical assays, which revealed that contacts between the BH and RuvC-lid (R964–E1020, E961–K1021) are only secondary to BH’s helical transition for orchestrating the conformational transitions required for double-strand cleavage and mismatch sensitivity. The wild-type protein with an intact conformational change in the BH is able to linearize (i.e., both NTS and TS cleavages) despite disrupting the BH-RuvC-lid interactions, with the only significant reduction being the second strand cleavage for MM13 substrate (Fig. 3e-f, Supplementary Figs. 17, 18, and Supplementary Table 11). Assessing mismatch sensitivity is also novel in this report compared to the previous work. • MD simulation results provide a mechanistic model for the structural and biochemical results. Correlation analyses reveal a reorganization of allosteric coupling between BH, RuvC-H1, and nucleic acids: from antagonistic DNA–crRNA interactions in S2, to DNA-dominated antagonism in S4b, and ultimately to strongly coordinated correlations with crRNA, DNA, RuvC-H1 and RuvC-lid in I4. Notably, this coordinated coupling is absent in the impaired BH variant, underscoring the requirement of BH’s loop-to-helical transition for establishing the allosteric network that licenses catalytic activation. To our knowledge, this is the first direct experimental and computational evidence implicating BH as the allosteric trigger to coordinate such diverse aspects (crRNA, DNA, REC2 docking, RuvC-H1 melting, and RuvC-lid opening) of the DNA cleavage competent state of Cas12a. • Alongside biochemical and computational analyses to investigate the role of bridge helix (BH) transitions in Cas12a activation, we also performed a comprehensive bioinformatics analysis to assess the structural conservation of BH–RuvC-helix 1 (H1) allosteric coupling across Cas12 and Cas9 protein families. We show conserved BH–RuvC-H1 mechanism in Cas12 subfamilies, while an absence of this coupling in Cas9 where REC lobe in inserted in between the BH and the RuvC helix. This, we believe, is the first report analyzing the similarities and differences of BH-mediated structural mechanisms of Cas9 and Cas12a. 15 3. Further, the two referenced manuscripts described above resolve structures of WT enzymes, whereas this manuscript focuses on a Cas12 variant with a modified bridge helix domain thought to slow kinetics of conformational transitions. While this mutant system may be descriptive of WT mechanisms, the lack of biochemical confirmation of their findings makes it challenging to distinguish if these lessons learned are broadly applicable or simply idiosyncrasies of an impaired system. To address this important point raised by the reviewer, we have added biochemical and MD simulation experiments in the revised manuscript. These results, along with the cryo-EM structures, demonstrate that the structural interactions that we identified are not idiosyncrasies, rather a potentially conserved mechanism in orthologs due to similar amino acids and conformational changes that are observed in the orthologs. Specifically: i. The BH arginines (e.g., R964 and R968 in FnoCas12a) are conserved (Supplementary Fig. 21) across Cas12a orthologs, which indicates that the interaction networks we describe, particularly those linking the BH and the RuvC-lid, are not specific to FnoCas12a but represent a general regulatory feature. ii. The biochemical experiments clearly demonstrate that BH’s loop-to-helical transition is the most important process in Cas12a’s activation rather than BH-RuvC interactions (please see details in the points for the previous comment). Our results implicate the following order of events in the activation process: BH’s helical transition, R-loop propagations past 16 nucleotides and REC2 docking by placement of W971 and opening of RuvC through BH-lid interactions. This clear distinction was possible through biochemical experiments that tested the variants with disrupted BH–RuvC-lid interactions in both FnoCas12aWT and FnoCas12aKD2P, as well as using matched and mismatched DNA substrates. Our results show that the impaired BH dynamics in FnoCas12aKD2P heighten mismatch sensitivity, reinforcing the fact that the BH’s conformational change is not only a checkpoint for activation for DNA cleavage but also a critical determinant of DNA cleavage fidelity, providing information that facilitate the improvement of gene editing accuracy. iii. MD simulation results show a shift in the correlated movements between BH–RuvC-H1, BH– RuvC-lid and BH–crRNA–DNA from anti-correlated to positively correlated interactions going from S2 to S4b to I4. This provides the first direct evidence of BH-mediated allostery in organizing the activated form of Cas12a for DNA cleavage. Together, these results show that, while the FnoCas12aKD2P variant allowed us to capture otherwise transient intermediates, the insights gained are not limited to this impaired system. Instead, the 16 combination of structural, biochemical, and computational analyses demonstrates that the BH’s helical transition and bending towards R-loop, RuvC-H1 melting, and BH–RuvC-lid interactions are general mechanistic features of Cas12a enzymes with direct implications on catalytic activation and RNA-DNA mismatch sensitivity. 4. While the authors have made efforts to biochemically characterize this specific mutant of FnoCas12a (Priya et al. 2021), and indeed some results from this previous manuscript strengthen claims made here, their previous work was largely focused on catalysis with little biochemical evidence to directly support the claims made in this manuscript which implicate the bridge helix in pre-catalysis conformational changes. More biochemistry is required here. We have addressed this concern by including biochemical characterization of several new variants that test the effect of interaction of BH and RuvC-lid residues in DNA cleavage mechanism. By comparing these variants in both FnoCas12aWT and FnoCas12aKD2P variant, our manuscript clearly establishes that the effect from BH’s helical transition and bending towards R-loop surpasses the requirement of BH–RuvC-lid interactions in orchestrating the changes needed for DNA cleavage (Figs. 3e-f, Supplementary Figs. 17, 18, and Supplementary Table 11). Please also see the detailed responses to the previous reviewer comments). 5. The figures in this manuscript are simplified to the point where it makes their interpretation challenging. Figures containing protein structures are under-labeled and stripped of context, requiring frequent reference to the manuscript, figure legend, and occasionally the provided structures, to properly understand. Increased labeling (as seen in figure 5B) and structural context callouts (as seen in figure 2A) would go a long way in increasing the clarity of the images to the reader. We appreciate the reviewer’s comment regarding figure clarity. To make the figures self-explanatory, we have revised the figures by: • Standardizing the color scheme for protein domains and RNA-DNA elements across all figures to minimize the need for cross-referencing • Adding labels and structural callouts (residues, domains, and regions of interest) to provide context within each panel • Including informative annotations within the figures themselves to highlight key conformational changes to reduce the reliance on figure legends 17 We believe that these changes will enable the figures to stand alone and improve readability. Minor Comments • The authors note on page 17 that their enzyme FnoCas12aKD2P is unable to perform trans-cleavage. Priya et al. 2021 demonstrates that while trans-cleavage is dramatically inhibited in FnoCas12aKD2P it does still occur if incubated for long enough – at least with M13 ssDNA. We appreciate the reviewer pointing out this discrepancy. We have reworded the text to clarify this by including discussion on data points from the previous publication. (Discussion section, page 21, lines 1-9, which is copied below). “In our previous work, we have also demonstrated that FnoCas12aKD2P has a lower trans-cleavage efficiency (0% substrate left for FnoCas12aWT at 5 minutes, compared to 40% remaining in FnoCas12aKD2P after two hours)14. Unlike FnoCas12aWT, which rapidly degraded substrates, FnoCas12aKD2P accumulated linear intermediates and failed to sustain a robust progressive trans-cleavage14. Additionally, FnoCas12aKD2P was unable to perform trans-cleavage when activated by a dsDNA14. The trans-cleavage occurs through keeping the RuvC domain “open” for non-specific ssDNA to access the active site24,53. These observations highlight that impairment of the BH’s helical transition likely blocks the prolonged activated form of the RuvC pocket, preventing trans-cleavage in FnoCas12aKD2P.” • While the bioinformatics was well done, I thought that, in line with my comments above, the associated figures highlighting structural similarities were unclear (though these were much better than previous figures). Further, the commentary on the bioinformatics portion of this manuscript was surface level and somewhat vague with little discussion beyond how they are structurally (and thus potentially mechanistically) similar. We suggest that the implications of this bioinformatic analysis be explored in the discussion section. For example, how frequently do these domain insertions occur across similar systems? We appreciate the reviewer's feedback on the bioinformatics analysis and figures. We performed further analysis by including the available structures of Cas9 subtypes (II-A, II-B and II-C) in our previous pairwise 3D structural comparison of the BH-RuvC domain of FnoCas12a and various Cas12 subtypes. This enabled us to make important observations regarding both the structural and mechanistic similarities and differences in the BH–RuvC-H1 regions of Cas12 subtypes as well as between Cas12 18 and Cas9 proteins. We have discussed differences in the primary and structural arrangement of Cas9 and Cas12 RuvC domain and how these structural arrangements are implicated in their conformational changes. We have also remade the figures highlighting structural similarities (Fig. 5b and Supplementary Figs. 22 and 23) for more clarity and have also included appropriate figures for the additional information on Cas9. • We found typos/grammatical errors in the manuscript, please proofread carefully. We appreciate the reviewer’s feedback and have conducted a thorough proofreading to ensure accuracy and clarity before submitting the revised version. Reviewer #3 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. We thank reviewer 3 for their assistance in the review process. 19 We thank the reviewers for their insightful comments, which were helpful in increasing the rigor of this manuscript. We have addressed these points in this revision and a point- by-point response to the questions are below, in blue. Reviewer #1: The authors have satisfactorily addressed all my previous comments. The revised manuscript is clearly improved in both presentation and data interpretation. The additional experiments and clarifications have strengthened the conclusions, and the responses were detailed and appropriate. I have no further concerns. We thank the reviewer for this feedback and are excited that the added experiments and clarifications support the conclusions. Reviewer #4 and co-reviewer #5 Comments: The molecular dynamics (MD) simulations complement the experimental structures by examining the conformational behavior of Cas12a across several experimentally defined functional states. The study focuses on how the bridge helix (BH) transition from a loop to a helix may contribute to the enzyme’s activation mechanism. The authors simulated four states: wild-type (I2 and I4) and mutant (S2 and S4b), each for 1 μs. Principal component analysis (PCA) revealed distinct conformational clusters for each state, which the authors interpret as a progression from early (S2/I2) to pre-catalytic (I4) conformations, with S4b representing an intermediate. Cross-correlation analyses were further used to probe potential allosteric coupling between the BH, RuvC-H1, RuvC-lid, and nucleic acids. According to the authors, these correlations become stronger in the active state, whereas the KD2P mutant shows disrupted and anti-correlated motions, which they propose explains its reduced catalytic efficiency. The molecular dynamics (MD) component of this study provides valuable mechanistic context to the experimental findings by exploring the conformational dynamics of Cas12a in different functional states. Overall, the MD protocol appears well-designed and appropriately detailed. The selection of trajectories and analyses is well aligned with the study’s stated goals. However, several points related to clarity, consistency, and methodological transparency could be improved to further strengthen the computational section. We thank the reviewers for the positive comments on how the MD simulations complement the structural and functional studies and help us solidify the molecular mechanisms. Below we list revisions that were made to improve clarity, consistency, and methodological transparency. Comment 1: The description of the cross-correlation analysis lacks sufficient methodological detail. It is not clear which software, module, or specific algorithm was used to compute the correlation matrices (e.g., CPPTRAJ, Bio3D, or an in-house script). 1 The reference frame, atom selection, and specific trajectory segments used for this analysis should also be specified for reproducibility. Additionally, it is not mentioned which frames of the MD trajectories were analyzed. Since the initial part of the simulation often represents equilibration rather than equilibrium dynamics, clarification is needed on whether only equilibrated frames (e.g., after RMSD or energy stabilization) were included, which is standard practice. This information is important to ensure that the reported correlations reflect genuine conformational dynamics rather than transient relaxation artifacts. Response 1: We thank the reviewers for the comment. We have modified the following paragraphs to provide more information on the MD analysis and to clarify our workflow. Specifically, in Supplemental Methods 3 (page S9, line 33 to page S10, line 6) and added Supplementary Figure 19 (page S41), we added a description of our use of CPPTRAJ, reference frame, atom selection, and trajectory segments in the cross-correlation analysis: “Cross-correlation analyses using CPPTRAJ were performed to delineate the allosteric control of BH’s loop-to-helical transition in other regions of the protein (RuvC-H1 and RuvC-lid) as well as with crRNA and DNA. As the RMSD values (Supplementary Fig. 19) indicated, all systems were equilibrated after 100 ns of MD simulation, which was further supported by the PCA analysis showing compact clusters for each trajectory (Supplementary Fig. 20). For the cross-correlation analysis, the reference was chosen to be the average structure of each MD simulation, and all carbon atoms of the protein along with the phosphorous atoms of the DNA/RNA were included in the analysis. The cross-correlation matrix was constructed using the last 900 ns of the MD trajectory, excluding the first 100 ns of equilibration.” Supplementary Figure 19: Root Mean Square Deviation (RMSD) of the simulation trajectory of FnoCas12aWT and FnoCas12aKD2P states. Colored lines represent the individual states: FnoCas12aKD2P-S2 (purple) and FnoCas12aKD2P-S4b (green) of FnoCas12aKD2P, and FnoCas12aWT-I2 (blue) and FnoCas12aWT-I4 (orange). The RMSDs were calculated by comparing each frame of the 1 μs MD trajectory to the initial structure of each system. 2 To further demonstrate the sampling convergence, below we color the PCA cluster for each system that is shown in Supplemental Figure 20a by the simulation time (ranging from blue to red as the simulation time increases). Fig. R1. Principal Component Analysis (PCA) of the simulation trajectories of FnoCas12aKD2P and FnoCas12aWT states. The analysis was performed on a total of 4 μs of concatenated MD trajectories comprising all four states. The color starts from blue and ends with red for each 1 μs trajectory. In the Methods section of the main text, we also added (page 29, lines 8-12): “Each system underwent 1 μs NVT simulation. The resulting trajectories, totaling 4 μs after including all the systems, were concatenated and analyzed using Principal Component Analysis (PCA). For the cross-correlation analysis, the first 100 ns of equilibration was excluded and only the last 900 ns of each trajectory were used. Both PCA and cross-correlation analyses were performed using CPPTRAJ⁶⁶.” Comment 2: In interpreting correlation results, correlation should not be equated with direct physical interaction. Statements such as “interaction network”, 'I4 state shows strong interactions with A3', and \"Lid also supports DNA positioning in I4\" could be softened to emphasize changes in correlated motion rather than binding. Response 2: We thank the reviewers for this comment. We have revised the text accordingly (Supplementary Methods 3, page S10, line 15-35): “Also, FnoCas12aKD2P-S4b displays a partial breakdown of this correlated motion network, with the emergence of a new network hotspot involving residues around 969 in BH–RuvC-H1 and 1017-1021 in RuvC-lid, indicating a possible structural rearrangement. These shifts suggest a progressive reorganization of the BH–RuvC-H1– RuvC-lid correlated motion network across the activation cycle. While FnoCas12aKD2P- S2 shows fewer contacts between these elements, FnoCas12aKD2P-S4b exhibits a noticeable increase, and FnoCas12aWT-I4 displays the most extensive correlated motion 3 network (Supplementary Fig. 21a). This trend indicates that FnoCas12aKD2P-S4b may represent a transitional state, where the coupling between BH–RuvC-H1 and the RuvC- lid is beginning to resemble that of the fully activated FnoCas12aWT-I4. Such a redistribution of contacts likely reflects functional remodeling and supports the idea that RuvC-lid engagement is strengthened as the complex progresses towards activation. The matrix cross-correlation analysis also shows a shift in the coordinated motions of RuvC-lid–crRNA in states S2 to S4b of FnoCas12aKD2P (Supplementary Fig. 21b). This is indicated by the absence of correlations (up to 0.44) between the RuvC-lid and crRNA in FnoCas12aKD2P-S2, highlighting that RuvC-lid is not coupled to crRNA positioning in this state (Supplementary Table 15). In FnoCas12aKD2P-S4b, the RuvC- lid–crRNA correlations become stronger (up to 0.46), with even more stronger correlations (over 0.55) for BH–RuvC-H1–crRNA as highlighted by residues 961-985 with A1 and G2 of crRNA (Supplementary Table 16). This suggests that BH–RuvC-H1 takes over crRNA coupling, pointing to a conformational shift in how the protein stabilizes the crRNA. In comparison, FnoCas12aWT-I44 shows strongly correlated motions of G5, U6, C7, and A8 of crRNA with residues in both RuvC-lid and BH–RuvC-H1 regions (Supplementary Table 17).” Comment 3: Consistency in the labeling of simulation states would improve readability. Throughout the MD section, state identifiers alternate between full notation (e.g., FnoCas12aKD2P–S2/S4 and FnoCas12aWT–I2/I4) and abbreviated forms (S2/S4, I2/I4). Standardizing this nomenclature across the text and figures would enhance clarity. Response 3: To address this, we have revised the MD section to match with the rest of the manuscript to ensure consistency in our nomenclature for the states. We have kept the protein name along with the specific state when we are referring to a selected structure. When we are referring to S2, S4b, and I4 as different conformational states towards reaching the activated complex, we have not used the protein name to give emphasis on the conformational state irrespective of the specific protein. Comment 4: Correlation values are not crystal clear in Fig. 4 and Fig. SF20. It is stated in the captions that “Correlation values range from +1 (red) to -1 (blue), with a visual cutoff applied for values above 0.4 and below -0.4 to enhance clarity”. This would normally be interpreted as the existence of a range of colors (and values) between red and blue, but as a matter of fact only red and blue are shown, no intermediate colors/values. Response 4: We thank the reviewers for the comment. The correlation values in the correlation plots (Fig. 4 and Supplementary Fig. 20) indeed ranges from +1 (red) to -1 (blue), with 0 (white) representing no correlation. The color intensity reflects the strength of the correlation, where saturated red or blue corresponds to the most positive or negative correlation, respectively. The correlations at the value of 0.4 appear as pink and -0.4 as light blue. Weaker correlations, with values above -0.4 and below 0.4, are not displayed in these plots. 4 We tried using another color other than white to represent the non-correlated values; however, adding another color made it difficult to understand the scheme. To enhance clarity, we have added a color bar and updated Figures 4c-e and Supplementary Figure 21 (page S43). Figure 4. Correlation and PCA analysis of MD simulations of FnoCas12aKD2P and FnoCas12aWT. a, Principal component analysis (PCA) of systems FnoCas12aKD2P states S2 (purple) and S4b (green), and FnoCas12aWT state I4 (orange). b, Close-up view of structural representation of the PC1 of Cas12a complex highlighting the motions of BH, RuvC-H1, and RuvC-lid between the different states. c-e, Correlation networks of internal dynamics derived from 1 μs of MD simulations for FnoCas12aKD2P-S2 in c, FnoCas12aKD2P-S4b in d, and FnoCas12aWT-I4 (PDB: 6GTG) in e. Circular plots represent correlations between four key regions: RuvC-H1 (purple), BH (pink), crRNA (orange), and DNA (blue). Correlation values range from +1 (red) to -1 (blue), with 0 (white) representing no correlation. The color intensity reflects the strength of the correlation, where saturated red or blue corresponds to the most positive or negative correlation, respectively. Visual cutoff was applied for values above -0.4 and below 0.4 to enhance clarity. Residues E961, R964, R968, K981, E1020, K1021, and N1022 are labeled in the correlation network. As the states progress from S2 to S4b to I4, there is an increase in the positively correlated motion between the BH, RuvC-lid, RuvC-H1, crRNA, and DNA. Due to the absence of sidechains on the experimental structure of 5 FnoCas12aWT-I2 (PDB: 6GTD), the cross-correlation network analysis was not performed for this state (please see Supplementary Methods 3 for details). “Supplementary Figure 21: Cross-correlation networks highlighting the strongest correlated motions in FnoCas12aKD2P-S2 and FnoCas12aKD2P-S4b and FnoCas12aWT-I44 states. a, Internal dynamic correlations between the BH–RuvC-H1 (residues 953-1000) and RuvC-lid (residues 1009-1021) domains, b, Cross-correlations between protein domains (BH–RuvC-H1 and RuvC-lid) and crRNA, highlighting just the crRNA correlations, and c, Cross-correlations between protein domains (BH–RuvC-H1 and RuvC-lid) and DNA, highlighting just the DNA correlations. Correlation values range from +1 (red) to -1 (blue), with 0 (white) representing no correlation. The color intensity reflects the strength of the correlation, where saturated red or blue corresponds to the most positive or negative correlation, respectively. Visual cutoff was applied for values above -0.4 and below 0.4 to enhance clarity. (related to Supplementary Tables 12-14)” 6 Below is an example for Figures 4c-e with a 0.1 cutoff and with residues E961, R964, R968, K981, E1020, K1021, and N1022 labeled in the correlation network. The figures show a similar pattern, but an easier interpretation is possible in Figures 4c-e by focusing on only the stronger correlations. Fig. R2. Cross-correlation networks highlighting the strongest correlated motions in S2 and S4b of FnoCas12aKD2P and FnoCas12aWT-I4. Replot of Figures 4c-e with a cut-off of 0.1 for positive and -0.1 for negative correlation. Circular plots represent correlations between five key regions: BH (pink), RuvC-H1 (purple), RuvC-lid (medium purple), crRNA (orange), and DNA (blue). Correlation values range from +1 (red) to -1 (blue), with 0 (white) representing no correlation. The color intensity reflects the strength of the correlation, where saturated red or blue corresponds to the most positive or negative correlation, respectively. Residues E961, R964, R968, K981, E1020, K1021, and N1022 are labeled in the correlation network. Comment 5: (Page 28) The following sentence could be revised for clarity: “Molecular dynamics (MD) simulations were performed using AMBER20 with appropriate force fields, including 1 μs production runs under NVT conditions and PCA on 4 μs of total simulation time using CPPTRAJ⁶⁶.” As currently written, the sentence combines multiple methodological details in a way that may be confusing. It could be clarified to specify that each system underwent a 1 μs production run under NVT conditions, and that the total 4 μs of trajectories were subsequently analyzed by principal component analysis (PCA) using CPPTRAJ. Response 5: We thank the reviewers for this comment. We modified the sentence to Main text, methods section, page 29, lines 8-12): “Each system underwent 1 μs NVT simulation. The resulting trajectories, totaling 4 μs with all the systems, were concatenated and analyzed using Principal Component Analysis (PCA). For the cross-correlation analysis, the first 100 ns of equilibration was excluded and only the last 900 ns of each trajectory were used. Both PCA and cross-correlation analyses were performed using CPPTRAJ⁶⁶.” Comment 6: (Page S8) In general, the simulation workflow is well-constructed, but some explanations could benefit from rephrasing for greater clarity. For example, the phrase “Pressure was maintained at 1 atm using the Berendsen barostat under periodic 7 boundary conditions during 200 ps” is vague and does not clearly indicate that this step corresponds to the pressure equilibration phase. It could be revised to more explicitly describe its role in the equilibration process. Response 6: We thank the reviewers for the suggestion. The sentence has been revised to better describe the NPT stage of the simulation workflow (Supplementary method 3, page S8 lines 27-32). “After thermalization, the system was equilibrated for 250 ps in the NPT ensemble at 1 bar using isotropic pressure scaling with the Berendsen barostat to adjust the density under periodic boundary conditions and to reach the target pressure. This was followed by a 1 μs NVT equilibration (100 ns) and production (900 ns), during which frames were recorded every 100 ps with a 1 fs timestep.” Comment 7: (Page S9) The statement “In PCA, the eigenvalues reflect the RMSD of Cα atoms” should be corrected for accuracy. In PCA of molecular dynamics simulations, the eigenvalues represent the variance (mean-square fluctuation) of the atomic coordinates along each principal component, rather than the root mean square deviation (RMSD). Response 7: We thank the reviewer for pointing out this issue. The sentence has been changed to (Supplementary method 3, page S9 lines 12-13): “In the PCA of MD simulations, the eigenvalues represent the variance (mean-square fluctuation) of the atomic coordinates along each principal component.” Comment 8: (Page S9) The sentence “In contrast, S4b represents a unique intermediate conformation between I2 and I4” seems redundant, as this point has already been discussed earlier. Response 8: We thank the reviewers for the suggestion. The sentence has been removed to avoid repetition (Supplementary Methods, page S9, lines 18-22). “The resulting 2D projection of PC1 and PC2 revealed distinct clustering for each state, indicating well-defined conformational ensembles (Supplementary Fig. 20a). Along the first principal component (PC1), a progression is observed from S2 and I2, through S4b, to I4, delineating a pathway consistent with the expected structural transition toward the pre-catalytic state of Cas12a (Supplementary Fig. 20b and Supplementary Video 2).” Comment 9: (Page S10) Furthermore, the reference to nucleotide G7 in the circular correlation plot appears inconsistent with the figure and should be verified. Response 9: We thank the reviewers for pointing this out. We verified the sequences for crRNA and DNA. The nucleotide from TS DNA should be dG-7 and the complementary RNA should be C7, as described in Supplementary Figure 8a and Supplementary table 19. The text and figures were revised to ensure consistency throughout the manuscript. 8 Comment 10: Finally, regarding data presentation and interpretation, the authors discuss correlations between the R-loop (crRNA–DNA hybrid) and DNA:", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their insightful comments, which were helpful in increasing the rigor of this manuscript. We have addressed these points in this revision and a point-by-point response to the questions are below, in blue.\nReviewer #1:\nThe authors have satisfactorily addressed all my previous comments. The revised manuscript is clearly improved in both presentation and data interpretation. The additional experiments and clarifications have strengthened the conclusions, and the responses were detailed and appropriate.\nI have no further concerns.\nWe thank the reviewer for this feedback and are excited that the added experiments and clarifications support the conclusions.\nReviewer #4 and co-reviewer #5\nComments:\nThe molecular dynamics (MD) simulations complement the experimental structures by examining the conformational behavior of Cas12a across several experimentally defined functional states. The study focuses on how the bridge helix (BH) transition from a loop to a helix may contribute to the enzyme’s activation mechanism. The authors simulated four states: wild-type (I2 and I4) and mutant (S2 and S4b), each for 1 μs. Principal component analysis (PCA) revealed distinct conformational clusters for each state, which the authors interpret as a progression from early (S2/I2) to pre-catalytic (I4) conformations, with S4b representing an intermediate. Cross-correlation analyses were further used to probe potential allosteric coupling between the BH, RuvC-H1, RuvC-lid, and nucleic acids. According to the authors, these correlations become stronger in the active state, whereas the KD2P mutant shows disrupted and anti-correlated motions, which they propose explains its reduced catalytic efficiency.\n[...]"}, {"round": 3, "reviewer_comments": {"reviewer_4": "We thank all the reviewers for their positive feedback about the significance of the work and their critical comments that have greatly helped us improve the quality of our manuscript and distinguish the contributions of this manuscript over the previously existing literature. A point-by-point response to the reviewer comments is below in blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript provides a detailed structural analysis of the bridge helix (BH) in Cas12a activation, offering insights into its role in allosteric regulation. The cryoEM data are well-analyzed, and the findings contribute meaningfully to the understanding of Cas12a’s conformational transitions. The study is of interest to researchers in CRISPR biology and genome editing. While the manuscript is generally well-prepared, some points require further clarification and discussion to strengthen the conclusions.\nWe thank the reviewer for the positive feedback about the significance of the work and the quality of cryo-EM data analysis and its contribution to understanding the role of BH in Cas12a’s activation. Points which needed further clarification are addressed in the revision as described below.\nMajor Comments\n1. The study suggests that BH’s loop-to-helical transition directly influences RuvC activation. However, it remains unclear whether this is a cause or a consequence of R-loop formation. Additional discussion, or reference to supporting biochemical evidence, would help clarify this point.\nTo clarify whether BH’s loop-to-helical transition is a cause or a consequence of R-loop formation and its influence on RuvC activation, we have conducted additional biochemical, structural, and computational analyzes. The results now clearly establish that BH’s loop-to-helical transition is a cause since without this transition, R-loop formation is inhibited mid-way through, and the formation of the helical structure of the hybrid is impaired. We also show that, with a variant able to undergo the loop-to-helical transition, BH–RuvC-lid interactions are only secondary as the variants maintain efficient on-target DNA cleavage without the BH–RuvC-lid interactions.\n1\nBiochemical Analysis:\nTo probe the allosteric mechanism, residues that mediate BH-lid interactions to convert the lid from a loop to a helical form at different stages of the activation process, thereby mediating the exposure of RuvC active site, were identified based on wild-type (FnoCas12aWT) and BH-variant (FnoCas12aKD2P) structures. Residues R964 of the BH and E1020 and K1021 of the RuvC-lid were substituted with alanine, along with a double variant E1020A/K1021A in both FnoCas12aWT and FnoCas12aKD2P backgrounds. In vitro plasmid DNA cleavage assays using both matched (MCH) and mismatched (MM) DNA substrates (MM2, PAM proximal mismatch and MM13, mid R-loop mismatch) were performed to assess the effects of the alanine substitutions. In the FnoCas12aWT background, single substitutions do not show prominent impairments, except for MM13-R964A combination, consistent with the stabilizing effect that the BH offers for the allosteric interactions. On the other hand, in the FnoCas12aKD2P background, where BH stability is impaired by the proline substitutions, the substitutions at R964 of the BH and the double variant (E1020/K1021) of the RuvC-lid, impacted both TS and NTS cleavages. In comparison, the only significant impact seen in FnoCas12aWT is the accumulation of nicked intermediate in R964A-MM13 combination (Figures 3e-f, Supplementary Figs.17,18, and Supplementary Table 11). These data clearly show that DNA cleavage is an additive process with combinations from effects from the BH’s loop-to-helical transition, opening of the RuvC active site by the helical transition of the lid, and the strength of complementary pairing between the RNA and DNA. Of these three, BH’s transition has the most significant contribution since in the wild-type backbone, all substitutions show minimal effect, except in R964-MM13, which indicates that the second contributor is the strength of complementary base pairing that is also dictated by the position of the mismatch between the RNA and DNA. The new results are presented in pages 12-15, with a summary in pages 14 (lines 23-26, and 15 (lines 1-10) which is copied below.\n“Our findings show that contacts between the BH and RuvC-lid (R964-E1020, E961-K1021) are only secondary to the BH’s helical transition for orchestrating the conformational transitions required for double-strand cleavage and mismatch sensitivity. The wild-type protein with an intact conformational change in the BH can linearize (i.e., both NTS and TS cleavages) despite disrupting the BH–RuvC-lid interactions, with the only significant reduction being in the second strand cleavage for MM13 substrate (Supplementary Fig. 17 and Supplementary Table 11). On the contrary, when BH’s sturdiness is restricted, as in the FnoCas12aKD2P variant, the enzyme becomes highly dependent on these interactions to provide RuvC active site access to ssDNA. In the FnoCas12aKD2P background, while R964A and E1020A/K1021A substitutions impair both strand cleavages, single substitutions (E1020A or K1021A) impede second-strand cleavage (Supplementary Fig. 18a and Supplementary Table 11). These\n2\nresults demonstrate that the BH’s loop-to-helical transition is an allosteric switch coupling R-loop propagation to catalysis and point to the importance of the helicity of the BH in assisting the passage of TS DNA into the RuvC active site pocket. BH, RuvC-H1, and RuvC-lid are potential engineering sites to reduce mismatch tolerance in Cas12a.”\nStructural Analysis:\nStructural analysis showed that R964 and R968 interact with nucleotides dG(-7), dT(-8), and U10 of crRNA/DNA in state I4 of FnoCas12aWT (Supplementary Fig.15), while the corresponding interaction is absent in FnoCas12aKD2P (a maximum of 11 base pairs is visible in State 4a). While position 11 of crRNA and TS DNA move closer to the BH in FnoCas12aWT, this interaction stays at around 20 Å FnoCas12aKD2P. Previous studies have shown that 16 nucleotides of base pairing of crRNA and DNA is a checkpoint for Cas12a activation (Stella et al., 2018 and Strohkendl et al., 2024). Thus, it appears that BH’s transition is essential for R-loop propagation, making BH’s transition the cause for R-loop propagation, especially past 10-nucleotides of base pairing. The bending of BH towards R-loop and latching into REC2 domain in parallel with the growing RNA-DNA hybrid appears to give the RNA-DNA hybrid a double-helical structure, implicating the importance of BH in R-loop propagation and stability. This stable configuration of the BH and the R-loop seems to facilitate a concomitant opening of the RuvC active pocket through RuvC-lid remodeling. These points have been added to the Discussion section (page 19 (lines 14-26), which is copied below).\n“In addition, the elongation of the BH due to the addition of the new helical turns and its bending towards the RNA-DNA hybrid appears to be critical in providing an anchorage for R-loop and to complete the RNA-DNA hybrid formation. In the FnoCas12aWT structures, states I2 and I320 have BH in a loop state prior to its the helical conversion. In these structures, BH is at a distance of 17 Å from the RNA-DNA hybrid with a total of 8-nucleotides of base pairing between the crRNA-guide and TS DNA. In comparison, in state I4, we see a fully formed and bent BH that is closer to the RNA-DNA hybrid (6 Å). In FnoCas12aKD2P, which is not capable of undergoing the loop-to-helical transition, we only see up to a maximum of 11-nucleotides of base pairing between the crRNA-guide and TS DNA, with a distance of ~20 Å from the BH. Interestingly, we also see stacking and distortion of bases towards the end of the R-loop, implicating that the strands are not aligned well with an impaired BH, hinting at the essentiality of the stability provided by the BH in these intermediary stages to drive R-loop propagation. The slowing down of the structural transitions is supported by the lower reaction kinetics of FnoCas12aKD2P when compared to FnoCas12aWT14..”\n3\nComputational Studies:\n⁠Our MD simulations indicate that the BH’s loop-to-helical transition actively shapes the conformational landscape of Cas12a by exerting allosteric influence over the RuvC-lid, crRNA, and DNA. Cross-correlation analyses show a shift from anti-correlated to positively correlated interactions across states, such as S2 to S4b to I4 (especially for nucleotides at position 6 and 8 of crRNA and -8 to -4 of DNA). This reorganization coincides only with the helical transition of the BH, which in turn facilitates the emergence of strong BH–crRNA, BH–DNA, and BH–RuvC-lid couplings in the pre-catalytic state. Overall, these data show that BH’s helical transition is an activating trigger regulating R-loop propagation and opening of RuvC active site pocket (Points have been added in the Results section, page 16 (lines 8-24), which is copied below, and Supplementary Methods 3).\n“To delineate potential allosteric communication between BH and nucleic acid, we performed correlation analyses of the residues E961, R964, R968, K981, E1020, K1021, and N1022 of the BH, RuvC-H1, and RuvC-lid, as well as nucleotides in crRNA and DNA (Fig. 4c-e). The complete correlation analysis is available in Supplementary Methods 3. Results reveal that BH–RuvC-H1 and RuvC-lid conformations exhibit strongly correlated motions with those of the crRNA and DNA, and that the motions are different between the different states. FnoCas12aKD2P S2 has fewer correlated motions (i.e., components move in the same direction) between the BH–RuvC-H1 and RuvC-lid, while there are several anti-correlated motions (i.e., components move in the opposite direction) between BH–RuvC-H1 and the RNA and DNA (Fig. 4c). In the intermediate state S4b, however, this correlation pattern shifts; there is an increase in the correlated motions between BH–RuvC-H1 and RuvC-lid and a few new motions with the crRNA, while there still is a substantial number of anti-correlated motions with crRNA and DNA (Fig. 4d). In I420 of FnoCas12aWT, we see only correlated motions between BH–RuvC-H1, RuvC-lid, crRNA, and DNA (Fig. 4e). Interestingly, some of the interactions with crRNA and DNA include nucleotides near the middle of the R-loop (~8) providing mechanistic clues on why mismatches between the crRNA and DNA in the R-loop away from the PAM are particularly sensitive in FnoCas12a. This reorganization of the correlation network in different states of the protein points to the pivotal role that the BH–RuvC-H1 transitions play in organizing the cleavage-competent form of Cas12a.”\n2. The manuscript suggests that the impaired BH prevents trans-cleavage, but no direct biochemical validation is provided. If prior studies have addressed this, a more explicit reference would be beneficial. Otherwise, acknowledging this limitation would improve clarity.\n4\nWe thank the reviewer for this helpful comment. While the current manuscript focuses on providing structural insights into the cis DNA cleavage mechanism of FnoCas12aKD2P, previous biochemical evidence from our group (Parameshwaran et al., 2021) reported that this variant has significantly reduced trans-cleavage efficiency compared to FnoCas12aWT). We now cite and discuss this study more explicitly in the discussion section to show that the BH transition and the corresponding allosteric activation of RNA/DNA/RuvC-lid are important for trans DNA cleavage as well. We have carefully worded the text to make it clearer to a reader that trans cleavage assay was done in our prior work and that the structural data provides the mechanistic framework that explains the previously observed defect (Points have been added in the Discussion section, page 21, lines 1-9, which is copied below).\n“In our previous work, we have also demonstrated that FnoCas12aKD2P has a lower trans-cleavage efficiency (0% substrate left for FnoCas12aWT at 5 minutes, compared to 40% remaining in FnoCas12aKD2P after two hours)14. Unlike FnoCas12aWT, which rapidly degraded substrates, FnoCas12aKD2P accumulated linear intermediates and failed to sustain a robust progressive trans-cleavage14. Additionally, FnoCas12aKD2P was unable to perform trans-cleavage when activated by a dsDNA14. The trans-cleavage occurs through keeping the RuvC domain “open” for non-specific ssDNA to access the active site24,53. These observations highlight that impairment of the BH’s helical transition likely blocks the prolonged activated form of the RuvC pocket, preventing trans-cleavage in FnoCas12aKD2P.”\n3. The role of BH in Cas12a is emphasized, but how this differs from Cas9 remains unclear. Since Cas9 does not require a major RuvC conformational shift, a brief discussion on this evolutionary divergence would add depth to the paper.\nWe thank the reviewer for this comment. It prompted us to include Cas9 proteins in our structural analyses. Further analyses by including the available structures of Cas9 subtypes (II-A, II-B and II-C) in using the pairwise 3D structural comparison of the BH-RuvC domain of FnoCas12a and various Cas12 subtypes (Fig. 5 and Supplementary Fig. 22) suggest that the Cas9 BH do not align completely with that of FnoCas12a, whereas a helix in the RuvC motif-II is structurally positioned in a similar arrangement as the RuvC-H1 of Cas12a. As the BH and the RuvC motif-II are placed farther away from each other due to the insertion of the REC lobe in between in Cas9, it may not be feasible to translate or couple the conformational changes in the BH to the RuvC motif-II’s helix or vice versa, similar to the coordinated transitions that we observe in Cas12a’s BH and H1 (Supplementary Fig. 23). In Cas12a, the BH and the RuvC-H1 of the RuvC motif-II are located close to each other both in the primary\n5\nsequence and the tertiary structure, facilitating such coordinated transitions. It should be noted that there is a loop to helical transition seen in the long BH of Cas9, which we have shown in our previous studies to possess an allosteric effect in DNA cleavage mechanism and RNA-DNA mismatch sensitivity (Babu et al., 2019, Babu et al., 2021 and Wu et al., 2025). Thus, while the BH’s helical transition and its allosteric effect on DNA cleavage seems to be a conserved mechanism in Cas9 and Cas12a, the structural features contributing to this allostery seem to be evolutionarily divergent. We have included these results and discussions in our revised manuscript [e.g., Results section, page 18 (lines 3-19), which is copied below].\n“Structural overlays of the BH and RuvC domain (motifs I, II, and III) of Cas9 (subtypes II-A, II-B, and II-C) with those of FnoCas12a show that the BH and a helix of the RuvC motif-II of Cas9 have a similar structural arrangement to that in Cas12a. While helix in the RuvC motif-II of Cas9 and Cas12a RuvC-H1 shows a better structural overlay, the BH of Cas9 is prominently longer and does not align with that of FnoCas12a (Fig. 5b, Supplementary Fig. 22-23). Another significant difference between Cas9 and Cas12 is the insertion of the REC lobe between BH and RuvC motif-II, compared to their direct connection in Cas12 families (Supplementary Fig. 23). This long separation can potentially disrupt coordinated conformational changes between BH and RuvC-H1 that are present in Cas12s. Within the Cas9, there is a loop-to-helical transition of the BH in response to RNA binding that has been demonstrated to impact DNA cleavage efficiency and DNA mismatch sensitivity6,11,13,32,33,52. These observations show that while the fundamental mechanism of loop-to-helical transition of the BH is conserved in Cas12a and Cas9, the mechanisms for coordinated conformational changes differ between them. While the RuvC-H1 of RuvC motif-II is co-opted by Cas12 families to undergo coupled transformations with the BH to orchestrate conformational changes needed for assembling the pre-catalytic state, such coupling is absent in Cas9 (Supplementary Fig. 23c). Thus, nature seems to have optimized Cas12a and Cas9 molecular mechanisms differently to attain the same goal of RNA-directed, sequence-specific, DNA cleavage.”\n4. Cas12a’s ability to recognize mismatches is only briefly mentioned. A more detailed discussion would be useful, especially given its implications for high-fidelity genome editing.\nWe thank the reviewer for raising this important point. To address this, we have expanded the discussion to highlight prior biochemical evidence that demonstrated FnoCas12aKD2P’s RNA-DNA mismatch sensitivity that was reported in our previous work (Parameshwaran et al., 2021). Additionally, we\n6\nincluded two selected DNA mismatches (MM2 and MM13, representing PAM-proximal and PAM-middle DNA mismatch) to characterize the on-target vs. off-target cleavage effects of the new variants that were created during the revision of this manuscript. These data show that combining BH and RuvC-lid amino acid substitutions exacerbate mismatch sensitivity and provides both structural and biochemical support for a BH-dependent checkpoint that enforces the DNA cleavage fidelity of Cas12a. [Discussion section, page 20 (lines 5-26), which is copied below].\n“In another role, the loop-to-helical transition of RuvC’s lid has been shown to play an important role in bringing the TS strand into the active site pocket for DNA cleavage21,27. The AsCas12a structures probing the TS strand entry mechanisms (PDB: 8SFP, 8SFQ and 8SFR21) also support these since the BH is extended towards the RNA-DNA hybrid, establishing interactions with the RuvC-lid helix, which leads to the exposure of the active site pocket in these structures. While we do not have structural features to support the TS entry mechanism due to the presence of partial DNA strands, BH’s helical transition appears to be critical in the passage and cleavage of the TS. This is supported by our previous work and biochemical assays reported in the present work. We observed a 6-fold reduction in TS compared to only a 3-fold reduction in NTS cleavage by FnoCas12aKD2P compared to that by FnoCas12aWT 14. Additionally, FnoCas12aKD2P discriminates the RNA-DNA mismatches by preventing TS cleavage since it was able to nick substrates with mismatches but not linearize them14. The efficiency to cleave mismatches is lower for all 20 nucleotides along the RNA-DNA hybrid, with mismatches at positions 12-17 of the target DNA producing higher accumulation of the nicked intermediate (61-77%) compared to that in FnoCas12aWT (3-19%)14. In this study, we observe that variants with disrupted BH–RuvC-lid interactions (R964A, E1020A/K1021A) in the FnoCas12aKD2P background exhibit pronounced reductions in cleavage of both TS and NTS, while the significant effect observed in FnoCas12aWT is the reduction in linearization and accumulation of nicked intermediate for MM13 substrate. Together, these results indicate that the BH’s loop-to-helical transition is the main driving force in R-loop propagation and the opening of the RuvC-lid in response to the total length of the RNA-DNA hybrid. Further studies are needed to find the mismatch-sensing mechanism of FnoCas12aKD2P, especially to understand why positions 12-18 of the RNA-DNA paired region is more sensitive compared to the rest of the R-loop positions.”\nMinor Comments\n1. In the abstract, the authors state, “Overall, our study enables the development of high-fidelity Cas12a and Cas9 variants by BH modifications.” However, the study does not provide direct data on BH\n7\nregulation in Cas9, making this claim difficult to support. Clarification or a more cautious phrasing would be advisable.\nWe thank the reviewer for pointing this out. To address this concern, we have modified the sentence in the abstract (page 2, lines 13-15) as follows:\n“Pairwise 3D structural comparison of the BH and RuvC of Cas12a and Cas9 provides insight into the diversity of BH-mediated DNA cleavage in these mechanistically similar enzymes”.\n2. Suggest the authors to enhance the figures clarity further. In many figures, the information is not sufficient. For example, Figure 2, what color indicates what component? What the state it is for Panel D to G. I noticed the authors mentioned it in the figure legend, however, this will affect the reading experience for the figure. If the author can label the component in the figure according to the color, that would be great. Many figures have this similar issue.\nWe thank the reviewer for this helpful suggestion. We agree that increasing figure clarity will improve readability of the manuscript. In the revised version, we have updated the figures by directly labeling components within the panels so that readers can readily identify which colors correspond to which domains or states without needing to refer to the legend.\nIn Figure 2, we now provide explicit in-panel labels for the BH, RuvC-H1, crRNA, and DNA across panels a-c, with consistent color-coding across all figures. We have also ensured that similar improvements are made to other multi-panel figures where components or states were previously shown in different colors. These revisions should substantially enhance the clarity and self-contained interpretability of the figures.\n3. In the Figure 2, the display of W971 is unclear. Because this is very important point, A clear cryoEM density of the residues to show the interaction distances would be helpful.\nWe thank the reviewer for this important suggestion. In the revised manuscript, we have improved the visualization of W971 by providing a clearer cryo-EM density map and explicitly displaying the interaction distances. To avoid overcrowding the main figure, these panels have been moved to the supplementary information (Supplementary Figs. 13a–d). This updated representation should allow readers to more clearly evaluate the density corresponding to W971 and assess its positioning relative to neighboring\n8\nresidues. We have shown states S1 and S4b of FnoCas12aKD2P in the figures since the density for the sidechain of residues W971 and Y579 are not visible in S2 and S4a and, in state S3, densities for both\nBH and REC2 domain are absent. These are described in the main text [Results section, pages 11, (lines 22-26), and 12 (lines 1-3) which is copied below] is as follows:\n“Another important conformational change towards activation of Cas12a is the insertion of the conserved W971 (residue immediately following the proline substitutions of FnoCas12aKD2P at 969 and 970) into the REC2 hydrophobic pocket (composed of amino acids Y579, K527, K524, R583 of FnoCas12aWT 12,16,22) to enable REC2 docking after hybrid formation. In FnoCas12aKD2P structures, we see insertion of W971 into the REC2 hydrophobic pocket in S1 and S4b (S2 and S4a do not show density in this area to determine sidechain position). However, the interacting distance of W971 with Y579 of the REC2 domain, which is at ~4 Å in FnoCas12aWT, remains farther in S1 and S4b (6 Å and 10 Å, respectively) (Supplementary Fig. 13).”\n9\nSupplementary Figure 13: BH transitions are important for REC2 domain docking. (a-d), The conserved residue W971, immediately following the K969 and D970 (969 and 970 were substituted with proline in FnoCas12aKD2P), docks into the REC2 hydrophobic pocket (composed of amino acids Y579, K527, K524, and R583) and interacts closely with Y579. Close-up view of the impaired BH of FnoCas12aKD2P impacting this interaction in a, S1 and b, S4b where W971 and Y579 are at a distance of 6 Å and 10 Å, respectively. Zoomed in view of electron density from the cryo-EM maps to show the sidechain density for residues W971 and Y579 in c, S1 and d, S4b. Close interaction between W971\n10\nand Y579 is observed in both a, binary (PDB: 5NG610) and b, full R-loop states (PDB: 6GTG4) in FnoCas12aWT. Two REC2 helices (V523-H538 and F565-T587, dodger blue), BH (pink), and RuvC-H1 (purple) are shown in this figure.\n11\nReviewer #2 (Remarks to the Author):\nGanguly et al. describes the potential importance of the bridge helix (BH) domain of the Cas12a from Francisella novicida (FnoCas12a) in mediating conformational state transitions throughout initial R-loop formation, implicating it as an allosteric activator of REC domain movement and in achievement of the enzyme’s final catalysis-primed conformation. The authors present a series of five (very nice, often high resolution) conformationally unique structures of a mutated FnoCas12aKD2P model system. These demonstrate the domain movements associated with the extension of the R-loop as it forms and how the CRISPR effector undergoes a large scale ‘opening’ in order to accommodate this extending heteroduplex. The structures are of varying quality, the authors interpreted the density reasonably.\nWe thank the reviewer for the positive feedback on the quality of the structures, the interpretation of the density, and the explanation about domain movements with respect to R-loop propagation.\nSuitability for Nature Communications\n1. Given the structural focus of this manuscript, I would recommend publication in a more suitably specialized journal (i.e. Structure). This manuscript is purely focused on structural biology and, as such, would be a better fit for journals more so catered to this focus. More biochemical analysis would be required for a broader audience.\nWe thank the reviewer for their critical comments. To improve the suitability of our manuscript to Nature Communications, in the revision, we have added detailed biochemical and computational analyses as well as deepened our bioinformatics analysis to include a comparison between the BHs of Cas12 and Cas9 families. The results further strengthen the structural insights from the cryo-EM data and provide experimental evidence for RuvC–BH and BH–RNA/DNA interactions. Finally, the computational results establish a cooperativity between all the three components (BH, RuvC-lid, RNA-DNA hybrid) providing the first direct evidence of BH’s allosteric mechanism in organizing a DNA cleavage competent state of Cas12a. The main changes are:\nBased on our structural analyses, we created single and double variants of FnoCas12aWT and FnoCas12aKD2P and performed plasmid cleavage assays with BH–RuvC-lid interface variants (R964A, E1020A, K1021A, E1020A/K1021A) in both FnoCas12aWT and FnoCas12aKD2P backgrounds. We tested the impact of these substitutions on mismatch discrimination in addition to on-target DNA cleavage to\n12\nsupport our structural interpretations. Details are present in Results section, page 12, lines 12 to page 15 line 10) with a summary in pages 14 (lines 23-26) and 15 (lines 1-10), which has been shown in page 2 of the rebuttal to address Major Comment 1 of Reviewer #1, biochemical analysis.\nIn support of our biochemical and structural analyses, our molecular dynamics simulations show that the loop to helical transition of the BH reshapes the conformational landscape of FnoCas12a by altering correlation networks between the BH, RuvC-lid, crRNA, and DNA. This highlights BH’s loop-to helical transition as the allosteric trigger needed to propagate R-loop beyond 15 nucleotides of pairing and the subsequent activation of Cas12a for DNA cleavage (i.e., loop to helical transition of RuvC-lid exposing the active site for DNA entry). Points have been added in the Results section, page 16 (lines 7-24), and Supplementary Methods 3, which has been shown in page 4 of the rebuttal to address Major Comment 1 of Reviewer #1, computational studies).\nOur bioinformatics analysis revealed similar structural organization of BH and the first helix of RuvC motif-II as in Cas12a across all Cas12 subtypes. Interestingly, our new analyses including Cas9 show that while there is a helix in the RuvC motif-II of Cas9 subtypes that is positioned in a similar arrangement as that of the RuvC-H1 of Cas12a’s RuvC-motif-II, the coordinated conformational changes between RuvC helix and BH is absent in apo, binary, and ternary structures of Cas9. The insertion of the large REC lobe between the BH and RuvC motif-II’s helix may deter such allostery between the BH and RuvC in Cas9. We point to our previous work on Cas9 BH’s conformational change that involves a loop-to-helical transition within the BH domain, alluding to the mechanistic similarities and structural differences of these divergent Cas proteins. Details are present in Results section, page17, line 3 to page18, line 19, a summary of which has been shown in pages 5-6 of the rebuttal to address Major Comment 3 of Reviewer #1.\nWe believe that incorporating these complementary approaches will make the study relevant to a broad readership interested not only in Cas12a structure but also in its biochemical mechanism, DNA cleavage fidelity, and potential applications in genome editing.\nMajor Comments\n2. The authors rightly bolster their results through comparison to similar structures in Stella et al., 2018, and Strohkendl et al., 2024, which describe conceptually similar findings focused on FnoCas12a and AsCas12a, respectively. However, it is unclear in this manuscript what is the new intellectual contribution\n13\naside from the role of this mutated bridge helix. The manuscript would benefit from a clearer presentation of what is new.\nWe thank the reviewer for this critical comment since that has helped us to differentiate our contributions and place them rightly among those from the previous literature. Here is a list of the main findings from our work and how it relates to previous literature.\n• The previous studies reported the structures of the wild-type (WT) Cas12a orthologs in several states. Our structures capture several conformational intermediates that are different than those that were captured in the previous work. These structures demonstrate how the inability of the BH’s loop-to-helical transition traps the protein in conformational intermediates compared to what was seen in WT under the normal conformational cascade. These structures directly connect the BH’s conformational change as a necessity for R-loop formation past the checkpoint (around 16 nucleotides), and how it is intimately related to RuvC-lid opening. The results also show that the structures of the BH-impaired variant are able to capture the earlier states (I1 to I3 of FnoCas12aWT) in the conformational cascade, but not the later ones, providing direct evidence that BH is an allosteric regulator for the conformational coordination towards the later stages of the activation process. Thus, while WT structures offer conformational changes needed for Cas12a activation, the importance of BH’s conformational change and its allosteric control is not discernible only by comparing the WT structures.\n• We also see slight differences in the BH’s conformational changes among the different orthologs. In FnoCas12a, the BH undergoes a loop-to-helical transition in response to R-loop propagation followed by the bending of the BH to latch onto the growing R-loop. However, in AsCas12a and LbCas12a, the BH remains elongated in the binary and the initial conformational states and bends toward the R-loop post 16 nucleotides of base pairing that is a conformational checkpoint in the activation process (Supplementary Fig. 24). Thus, the BH’s loop-to-helical transition in FnoCas12a appears to serve as an additional structural sensor for RNA-DNA hybrid propagation, melting of RuvC’s H1, and opening of RuvC-lid that are all needed for catalytic activation.\n• The functionally important W971 that inserts into the REC2 domain in the later stages of catalytic activation is the residue right adjacent to the BH, located in-between the BH and RuvC H1. Our structures show that an impaired loop-to-helical conformational change of the BH prevents\n14\ninsertion of W971 to REC2’s hydrophobic pocket that implicates that proper REC2 docking is facilitated by BH’s conformational change.\n• We validated our structural findings through biochemical assays, which revealed that contacts between the BH and RuvC-lid (R964–E1020, E961–K1021) are only secondary to BH’s helical transition for orchestrating the conformational transitions required for double-strand cleavage and mismatch sensitivity. The wild-type protein with an intact conformational change in the BH is able to linearize (i.e., both NTS and TS cleavages) despite disrupting the BH-RuvC-lid interactions, with the only significant reduction being the second strand cleavage for MM13 substrate (Supplementary Fig. 17 and Supplementary Table 11). On the contrary, with an impaired BH, disrupting the BH–RuvC-lid interactions exacerbate the DNA cleavage deficiency in FnoCas12aKD2P backbone (Fig. 3e-f, Supplementary Figs. 17, 18, and Supplementary Table 11). Assessing mismatch sensitivity is also novel in this report compared to the previous work.\n• MD simulation results provide a mechanistic model for the structural and biochemical results. Correlation analyses reveal a reorganization of allosteric coupling between BH, RuvC-H1, and nucleic acids: from antagonistic DNA–crRNA interactions in S2, to DNA-dominated antagonism in S4b, and ultimately to strongly coordinated correlations with crRNA, DNA, RuvC-H1 and RuvC-lid in I4. Notably, this coordinated coupling is absent in the impaired BH variant, underscoring the requirement of BH’s loop-to-helical transition for establishing the allosteric network that licenses catalytic activation. To our knowledge, this is the first direct experimental and computational evidence implicating BH as the allosteric trigger to coordinate such diverse aspects (crRNA, DNA, REC2 docking, RuvC-H1 melting, and RuvC-lid opening) of the DNA cleavage competent state of Cas12a.\n• Alongside biochemical and computational analyses to investigate the role of bridge helix (BH) transitions in Cas12a activation, we also performed a comprehensive bioinformatics analysis to assess the structural conservation of BH–RuvC-helix 1 (H1) allosteric coupling across Cas12 and Cas9 protein families. We show conserved BH–RuvC-H1 mechanism in Cas12 subfamilies, while an absence of this coupling in Cas9 where REC lobe in inserted in between the BH and the RuvC helix. This, we believe, is the first report analyzing the similarities and differences of BH-mediated structural mechanisms of Cas9 and Cas12a.\n15\n3. Further, the two referenced manuscripts described above resolve structures of WT enzymes, whereas this manuscript focuses on a Cas12 variant with a modified bridge helix domain thought to slow kinetics of conformational transitions. While this mutant system may be descriptive of WT mechanisms, the lack of biochemical confirmation of their findings makes it challenging to distinguish if these lessons learned are broadly applicable or simply idiosyncrasies of an impaired system.\nTo address this important point raised by the reviewer, we have added biochemical and MD simulation experiments in the revised manuscript. These results, along with the cryo-EM structures, demonstrate that the structural interactions that we identified are not idiosyncrasies, rather a potentially conserved mechanism in orthologs due to similar amino acids and conformational changes that are observed in the orthologs. Specifically:\ni. The BH arginines (e.g., R964 and R968 in FnoCas12a) are conserved (Supplementary Fig. 21) across Cas12a orthologs, which indicates that the interaction networks we describe, particularly those linking the BH and the RuvC-lid, are not specific to FnoCas12a but represent a general regulatory feature.\nii. The biochemical experiments clearly demonstrate that BH’s loop-to-helical transition is the most important process in Cas12a’s activation rather than BH-RuvC interactions (please see details in the points for the previous comment). Our results implicate the following order of events in the activation process: BH’s helical transition, R-loop propagations past 16 nucleotides and REC2 docking by placement of W971 and opening of RuvC through BH-lid interactions. This clear distinction was possible through biochemical experiments that tested the variants with disrupted BH–RuvC-lid interactions in both FnoCas12aWT and FnoCas12aKD2P, as well as using matched and mismatched DNA substrates. Our results show that the impaired BH dynamics in FnoCas12aKD2P heighten mismatch sensitivity, reinforcing the fact that the BH’s conformational change is not only a checkpoint for activation for DNA cleavage but also a critical determinant of DNA cleavage fidelity, providing information that facilitate the improvement of gene editing accuracy.\niii. MD simulation results show a shift in the correlated movements between BH–RuvC-H1, BH–RuvC-lid and BH–crRNA–DNA from anti-correlated to positively correlated interactions going from S2 to S4b to I4. This provides the first direct evidence of BH-mediated allostery in organizing the activated form of Cas12a for DNA cleavage.\nTogether, these results show that, while the FnoCas12aKD2P variant allowed us to capture otherwise transient intermediates, the insights gained are not limited to this impaired system. Instead, the\n16\ncombination of structural, biochemical, and computational analyses demonstrates that the BH’s helical transition and bending towards R-loop, RuvC-H1 melting, and BH–RuvC-lid interactions are general mechanistic features of Cas12a enzymes with direct implications on catalytic activation and RNA-DNA mismatch sensitivity.\n4. While the authors have made efforts to biochemically characterize this specific mutant of FnoCas12a (Priya et al. 2021), and indeed some results from this previous manuscript strengthen claims made here, their previous work was largely focused on catalysis with little biochemical evidence to directly support the claims made in this manuscript which implicate the bridge helix in pre-catalysis conformational changes. More biochemistry is required here.\nWe have addressed this concern by including biochemical characterization of several new variants that test the effect of interaction of BH and RuvC-lid residues in DNA cleavage mechanism. By comparing these variants in both FnoCas12aWT and FnoCas12aKD2P variant, our manuscript clearly establishes that the effect from BH’s helical transition and bending towards R-loop surpasses the requirement of BH–RuvC-lid interactions in orchestrating the changes needed for DNA cleavage (Figs. 3e-f, Supplementary Figs. 17, 18, and Supplementary Table 11). Please also see the detailed responses to the previous reviewer comments).\n5. The figures in this manuscript are simplified to the point where it makes their interpretation challenging. Figures containing protein structures are under-labeled and stripped of context, requiring frequent reference to the manuscript, figure legend, and occasionally the provided structures, to properly understand. Increased labeling (as seen in figure 5B) and structural context callouts (as seen in figure 2A) would go a long way in increasing the clarity of the images to the reader.\nWe appreciate the reviewer’s comment regarding figure clarity. To make the figures self-explanatory, we have revised the figures by:\n• Standardizing the color scheme for protein domains and RNA-DNA elements across all figures to minimize the need for cross-referencing\n• Adding labels and structural callouts (residues, domains, and regions of interest) to provide context within each panel\n• Including informative annotations within the figures themselves to highlight key conformational\nchanges to reduce the reliance on figure legends\n17\nWe believe that these changes will enable the figures to stand alone and improve readability.\nMinor Comments\n• The authors note on page 17 that their enzyme FnoCas12aKD2P is unable to perform trans-cleavage. Priya et al. 2021 demonstrates that while trans-cleavage is dramatically inhibited in FnoCas12aKD2P it does still occur if incubated for long enough – at least with M13 ssDNA.\nWe appreciate the reviewer pointing out this discrepancy. We have reworded the text to clarify this by including discussion on data points from the previous publication. (Discussion section, page 21, lines 1-9, which is copied below).\n“In our previous work, we have also demonstrated that FnoCas12aKD2P has a lower trans-cleavage efficiency (0% substrate left for FnoCas12aWT at 5 minutes, compared to 40% remaining in FnoCas12aKD2P after two hours)14. Unlike FnoCas12aWT, which rapidly degraded substrates, FnoCas12aKD2P accumulated linear intermediates and failed to sustain a robust progressive trans-cleavage14. Additionally, FnoCas12aKD2P was unable to perform trans-cleavage when activated by a dsDNA14. The trans-cleavage occurs through keeping the RuvC domain “open” for non-specific ssDNA to access the active site24,53. These observations highlight that impairment of the BH’s helical transition likely blocks the prolonged activated form of the RuvC pocket, preventing trans-cleavage in FnoCas12aKD2P.”\n• While the bioinformatics was well done, I thought that, in line with my comments above, the associated figures highlighting structural similarities were unclear (though these were much better than previous figures). Further, the commentary on the bioinformatics portion of this manuscript was surface level and somewhat vague with little discussion beyond how they are structurally (and thus potentially mechanistically) similar. We suggest that the implications of this bioinformatic analysis be explored in the discussion section. For example, how frequently do these domain insertions occur across similar systems?\nWe appreciate the reviewer's feedback on the bioinformatics analysis and figures. We performed further analysis by including the available structures of Cas9 subtypes (II-A, II-B and II-C) in our previous pairwise 3D structural comparison of the BH-RuvC domain of FnoCas12a and various Cas12 subtypes. This enabled us to make important observations regarding both the structural and mechanistic similarities and differences in the BH–RuvC-H1 regions of Cas12 subtypes as well as between Cas12\n18\nand Cas9 proteins. We have discussed differences in the primary and structural arrangement of Cas9 and Cas12 RuvC domain and how these structural arrangements are implicated in their conformational changes. We have also remade the figures highlighting structural similarities (Fig. 5b and Supplementary Figs. 22 and 23) for more clarity and have also included appropriate figures for the additional information on Cas9.\n• We found typos/grammatical errors in the manuscript, please proofread carefully.\nWe appreciate the reviewer’s feedback and have conducted a thorough proofreading to ensure accuracy and clarity before submitting the revised version.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank reviewer 3 for their assistance in the review process.\n19\nWe thank the reviewers for their insightful comments, which were helpful in increasing\nthe rigor of this manuscript. We have addressed these points in this revision and a point-\nby-point response to the questions are below, in blue.\nReviewer #1:\nThe authors have satisfactorily addressed all my previous comments. The revised\nmanuscript is clearly improved in both presentation and data interpretation. The\nadditional experiments and clarifications have strengthened the conclusions, and the\nresponses were detailed and appropriate.\nI have no further concerns.\nWe thank the reviewer for this feedback and are excited that the added experiments and\nclarifications support the conclusions.\nReviewer #4 and co-reviewer #5\nComments:\nThe molecular dynamics (MD) simulations complement the experimental structures by\nexamining the conformational behavior of Cas12a across several experimentally defined\nfunctional states. The study focuses on how the bridge helix (BH) transition from a loop\nto a helix may contribute to the enzyme’s activation mechanism. The authors simulated\nfour states: wild-type (I2 and I4) and mutant (S2 and S4b), each for 1 μs. Principal\ncomponent analysis (PCA) revealed distinct conformational clusters for each state,\nwhich the authors interpret as a progression from early (S2/I2) to pre-catalytic (I4)\nconformations, with S4b representing an intermediate. Cross-correlation analyses were\nfurther used to probe potential allosteric coupling between the BH, RuvC-H1, RuvC-lid,\nand nucleic acids. According to the authors, these correlations become stronger in the\nactive state, whereas the KD2P mutant shows disrupted and anti-correlated motions,\nwhich they propose explains its reduced catalytic efficiency.\nThe molecular dynamics (MD) component of this study provides valuable mechanistic\ncontext to the experimental findings by exploring the conformational dynamics of Cas12a\nin different functional states. Overall, the MD protocol appears well-designed and\nappropriately detailed. The selection of trajectories and analyses is well aligned with the\nstudy’s stated goals. However, several points related to clarity, consistency, and\nmethodological transparency could be improved to further strengthen the computational\nsection.\nWe thank the reviewers for the positive comments on how the MD simulations\ncomplement the structural and functional studies and help us solidify the molecular\nmechanisms. Below we list revisions that were made to improve clarity, consistency, and\nmethodological transparency.\nComment 1: The description of the cross-correlation analysis lacks sufficient\nmethodological detail. It is not clear which software, module, or specific algorithm was\nused to compute the correlation matrices (e.g., CPPTRAJ, Bio3D, or an in-house script).\n1\nThe reference frame, atom selection, and specific trajectory segments used for this\nanalysis should also be specified for reproducibility. Additionally, it is not mentioned which\nframes of the MD trajectories were analyzed. Since the initial part of the simulation often\nrepresents equilibration rather than equilibrium dynamics, clarification is needed on\nwhether only equilibrated frames (e.g., after RMSD or energy stabilization) were\nincluded, which is standard practice. This information is important to ensure that the\nreported correlations reflect genuine conformational dynamics rather than transient\nrelaxation artifacts.\nResponse 1:\nWe thank the reviewers for the comment. We have modified the following paragraphs to\nprovide more information on the MD analysis and to clarify our workflow. Specifically, in\nSupplemental Methods 3 (page S9, line 33 to page S10, line 6) and added\nSupplementary Figure 19 (page S41), we added a description of our use of CPPTRAJ,\nreference frame, atom selection, and trajectory segments in the cross-correlation\nanalysis:\n“Cross-correlation analyses using CPPTRAJ were performed to delineate the allosteric\ncontrol of BH’s loop-to-helical transition in other regions of the protein (RuvC-H1 and\nRuvC-lid) as well as with crRNA and DNA. As the RMSD values (Supplementary Fig.\n19) indicated, all systems were equilibrated after 100 ns of MD simulation, which was\nfurther supported by the PCA analysis showing compact clusters for each trajectory\n(Supplementary Fig. 20). For the cross-correlation analysis, the reference was chosen\nto be the average structure of each MD simulation, and all carbon atoms of the protein\nalong with the phosphorous atoms of the DNA/RNA were included in the analysis. The\ncross-correlation matrix was constructed using the last 900 ns of the MD trajectory,\nexcluding the first 100 ns of equilibration.”\nSupplementary Figure 19: Root Mean Square Deviation (RMSD) of the simulation\ntrajectory of FnoCas12aWT and FnoCas12aKD2P states. Colored lines represent the\nindividual states: FnoCas12aKD2P-S2 (purple) and FnoCas12aKD2P-S4b (green) of\nFnoCas12aKD2P, and FnoCas12aWT-I2 (blue) and FnoCas12aWT-I4 (orange). The RMSDs\nwere calculated by comparing each frame of the 1 μs MD trajectory to the initial structure\nof each system.\n2\nTo further demonstrate the sampling convergence, below we color the PCA cluster for\neach system that is shown in Supplemental Figure 20a by the simulation time (ranging\nfrom blue to red as the simulation time increases).\nFig. R1. Principal Component Analysis (PCA) of the simulation trajectories of\nFnoCas12aKD2P and FnoCas12aWT states. The analysis was performed on a total of 4 μs\nof concatenated MD trajectories comprising all four states. The color starts from blue and\nends with red for each 1 μs trajectory.\nIn the Methods section of the main text, we also added (page 29, lines 8-12):\n“Each system underwent 1 μs NVT simulation. The resulting trajectories, totaling 4 μs\nafter including all the systems, were concatenated and analyzed using Principal\nComponent Analysis (PCA). For the cross-correlation analysis, the first 100 ns of\nequilibration was excluded and only the last 900 ns of each trajectory were used. Both\nPCA and cross-correlation analyses were performed using CPPTRAJ⁶⁶.”\nComment 2: In interpreting correlation results, correlation should not be equated with\ndirect physical interaction. Statements such as “interaction network”, 'I4 state shows\nstrong interactions with A3', and \"Lid also supports DNA positioning in I4\" could be\nsoftened to emphasize changes in correlated motion rather than binding.\nResponse 2: We thank the reviewers for this comment. We have revised the text\naccordingly (Supplementary Methods 3", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their insightful comments. We have addressed each point in the revised manuscript, and our responses are provided below in blue.\nReviewer #4:\nWe appreciate that the authors did a thorough revision and provided detailed responses to our comments. We have evaluated the rebuttal and the revised manuscript. Most issues have been addressed satisfactorily; however, a few points require additional clarification and revision as outlined below.\n[...]"}]

Decision letter