CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain

Domain: neuroscience
Published: 2026-01-24
Editorial outcome: minor_revision

Abstract
Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.

Introduction
Several prokaryotes have adaptive immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR) and genes expressing CRISPR-associated (Cas) proteins1. Due to their ever-growing functional versatility, these systems are being repurposed for a series of biotechnological applications, including gene editing, regulation, and detection2. Indeed, they have become essential tools for precise gene editing, enabling targeted modifications in the genomes of different organisms, including humans3. Increasing efforts have significantly enhanced the precision of the process to achieve therapeutic applications4. CRISPR-Cas systems have also allowed for the regulation of gene expression by activating or repressing specific genes without altering the DNA sequence5. RNA-guided dead Cas proteins offer opportunities for circuit engineering and gene function screening6. Moreover, the trans-cleavage activity upon nucleic acid targeting of both Cas12 and Cas13 has been instrumental to boost the field of CRISPR diagnostics7,8, allowing the sensitive detection of pathogens and genetic mutations.

Recently, the trans-cleavage activity of Cas9 has been disclosed over poly(T) single-stranded DNA (ssDNA) molecules9. Cas9 can also process C-rich ssDNA substrates and even small RNA molecules. This finding contrasts with a long-held opposite view based on structural considerations. Cas9 is a large multidomain protein that contains two endonuclease domains, RuvC and HNH, along with a protospacer adjacent motif (PAM)-recognizing domain10, which introduces double-stranded breaks in DNA at specific sites in an RNA-guided manner. Such a function of Cas9 is accomplished by the RuvC domain9 and apparently enhanced when the nuclease is guided in its native form with two RNA molecules, i.e., with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Nevertheless, the sequence and structural determinants of this collateral activity are not fully appreciated. In this work, we further investigate the trans-cleavage activity of Cas9 through a series of biochemical assays involving different targets, guides, and nucleases. We characterize how function is modulated by fundamental structural variations in nucleic acids and changes in protein activity.

Results
We considered a set of nucleic acid targets in the form of short double-stranded DNA (dsDNA) containing the canonical PAM (NGG), along with the corresponding crRNAs with a 20–22 nt spacer (more in detail, we used targets with a 20 bp stretch from the 5′ end to the PAM). We also considered the corresponding single-guide RNAs (sgRNAs) for a comparative assessment. These five systems had different arbitrary sequences and served to evaluate the impact of structural variations (Fig. 1A; see also Supplementary Fig. 1A). In this context, there is no stretch of hybridized DNA beyond the PAM-distal region of the protospacer that completes the formation of the R-loop upon targeting with the CRISPR-Cas9 complex11, thereby leading to an unflanked R-loop configuration. Using fluorogenic dsDNA molecules and a fluorogenic 18 nt poly(T) ssDNA probe, we scrutinized the cis-cleavage (measuring the DNA targeting ability as a proxy) and trans-cleavage activities of the Streptococcus pyogenes Cas9 in each case (Fig. 1B, C). According to our results, both functions are not aligned, as e.g., a high targeting ability led only to a moderate trans-cleavage activity with both the crRNA-tracrRNA and sgRNA in the case of system #2 or with the sgRNA in the case of system #5. Alternatively, a low targeting ability resulted in a high trans-cleavage activity with the crRNA-tracrRNA in the case of system #4. We also observed that while dsDNA targeting is higher with either the crRNA-tracrRNA or sgRNA, depending on the system (e.g., #1 vs. #4; note, however, it is quite similar with both guides in #3), ssDNA trans-cleavage is always higher or at least equal when Cas9 is guided in its native form (Fig. 1B, C). In systems #3, #4, and #5, no post-PAM extensions occurred, but we found little effect of this variation in our reaction conditions (Supplementary Fig. 1B, C). Moreover, some basal trans-cleavage activity (i.e., in the absence of target DNA) was noticed (Supplementary Fig. 1D), as it has also been observed with Cas12 nucleases12. These results are consistent with the reported catalytic feature and indicate that the magnitude of both cis- and trans-actions varies systematically with sequence composition.

To distill a clear structural determinant, we next considered longer dsDNA molecules to target (35–36 bp stretch from the 5′ end to the PAM). In this case, the DNA region flanking the resulting R-loop in the RNA 5′ side remains hybridized if using a crRNA or sgRNA with a 20–22 nt spacer, leading to a canonical (flanked) R-loop configuration (Fig. 1D). Intriguingly, we found a systematic substantial reduction in trans-cleavage activity when using long dsDNA inputs compared to the signals obtained with the shorter inputs (Fig. 1E, F; see also absolute fluorescence levels in Supplementary Fig. 2A, B). In the case of system #4, e.g., the reduction was 90% by guiding Cas9 with the crRNA-tracrRNA and 81% with the sgRNA. Although the absolute collateral activities differed, crRNA-tracrRNAs and sgRNAs showed comparable signals when normalized to their respective basal levels. Here, cis-cleavage was confirmed by gel electrophoresis (Supplementary Fig. 2C). We then found that by targeting long ssDNA molecules with crRNA-tracrRNAs (in a PAM-independent way)13 the trans-cleavage activity was almost recovered, except in one case in which this process was not efficient (#5). Similar results were obtained with sgRNAs, although that recovery was not observed in the case of systems #3 and #5. Using a fluorogenic 13 nt poly(T) ssDNA probe, we also observed a decline in its processing when the R-loop was flanked (Supplementary Fig. 2D). Yet, it was noteworthy that the higher trans-cleavage activity was observed in the case of systems #1 and #4 with long ssDNA inputs. By contrast, no apparent trans-cleavage activity was observed with a fluorogenic 18 nt poly(A) ssDNA probe (Supplementary Fig. 2E). Together, our data entail that a duplex-flanked R-loop in the RNA 5′ side hinder the collateral catalytic activity of Cas9.

We assayed crRNAs with imperfect spacers for systems #1 and #2, introducing mismatches at various positions to probe R-loop formation and downstream cleavage activities. Single-nucleotide mismatches were introduced at positions 5, 8, 13, and 19. A two-nucleotide mismatch at positions 5, 6 and a three-nucleotide mismatch at positions 4, 5, 6 were considered too. Cas9 tolerated single-nucleotide mismatches across all positions with respect to cis-cleavage (Fig. 2A, C). Yet, collateral activity was more sensitive and sequence-dependent; it was substantially reduced by the mismatch at position 5 in system #1 and by the mismatch at position 13 in system #2 (Fig. 2B, D; see also Supplementary Fig. 2F). Different basal activities were also noticed for each crRNA sequence. Furthermore, we observed that while the three-nucleotide mismatch impeded the formation of the complete R-loop in both systems, the two-nucleotide mismatch only did so in system #2 (Fig. 2A, C). These high-order mismatches nonetheless avoided a significant increase in trans-cleavage activity in both systems (Fig. 2B, D). These data underscore complexities associated with responsiveness and fidelity in the CRISPR-Cas9 system14.

To further study the dynamics of the response, we monitored over time the fluorescence signals associated with dsDNA targeting and subsequent ssDNA trans-cleavage (for system #1 with the crRNA-tracrRNA). Cis-targeting was fast, with a mean reaction time of ~7 min, but a delay of ~10 min was found in trans-cleavage together with a slower reaction rate (Fig. 2E). Of note, ssDNA collateral catalysis was completely abolished, even the basal activity of the ribonucleoprotein, using an extra long dsDNA target (49 bp stretch from the 5′ end to the PAM), in agreement with the formation of a more stable flanked R-loop. Examination of trans-cleavage activity for increasingly long dsDNA targets was in tune (Supplementary Fig. 2G). Indeed, this behavior was reproduced when targeting a plasmid (Fig. 2F). This suggests that this feature might play a limited role in the natural in vivo context, especially considering the lack of apparent impact on immunity in the case of Cas1215.

Subsequently, we reasoned that the collateral activity of Cas9 could be modulated in a quantitative manner through R-loop reconfigurations. Indeed, we observed that the longer the DNA stretch from the 5′ end to the PAM, the lower the trans-cleavage activity (Fig. 3A; Cas9 guided by crRNA-tracrRNAs, relative to system #1). More in detail, such activity correlated with the free energy of the duplex flanking the R-loop in the RNA 5′ side calculated from an empirical thermodynamic model16. Then, we analyzed the effect of simultaneous crRNA spacer elongations to maintain an unflanked R-loop in the RNA 5′ side. Surprisingly, we found that poly(T) probe processing was disrupted when using these longer crRNAs (Fig. 3B; see also Supplementary Fig. 3). To shed light, we created a series of crRNAs with strict spacer lengths (including the GR nucleotides in the 5′ end required for in vitro transcription with a T7 RNA polymerase). Elongating the crRNA spacer roughly maintained the DNA binding capacity (Fig. 3C); a feature that has already been exploited for nucleic acid detection17. However, trans-cleavage activity decreased with spacer length following an early linear trend (Fig. 3D). In particular, the reduction was 49% with a spacer of strict 22 nt and 79% with 24 nt. We also observed a slight increase in trans-cleavage activity with respect to the canonical case using a crRNA with a 19 nt spacer. Here, we confirmed by gel electrophoresis that Cas9 was competent for cis-cleavage with an elongated crRNA (Fig. 3E), although previous results have also suggested a reduction in on-target cleavage activity by modifying the 5′ end of the sgRNA18. Thus, our data demonstrate that spacer length is a critical and predominant variable to quantitatively control the collateral activity of Cas9, with progressive spacer elongation markedly suppressing trans-cleavage while preserving target binding and cis-cleavage competence.

To place our findings in an applied context, we conducted nucleic acid detection assays. To this end, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)19 was considered as the nucleic acid of interest (Fig. 4A). Using custom primers targeting the E gene, dsDNA amplicons containing the PAM sequence AGG were generated isothermally by reverse transcription recombinase polymerase amplification (RT-RPA)20, and then CRISPR-Cas9 reactions were run with a suitable crRNA-tracrRNA (20 nt spacer; Fig. 4B). Two different amplicons were generated, one with a short pre-PAM region of 28 bp (105 bp in total) and another with a long pre-PAM region of 111 bp (188 bp in total). We only obtained a differential fluorescence readout in the case of the amplicon with a short pre-PAM region, which leads to an unflanked R-loop configuration after targeting favored by temperature (Fig. 4C). The inability to detect the longer amplicon is in tune with the fact that a highly stable R-loop-flanking duplex in the RNA 5′ side limits the collateral activity of Cas9 (see also Supplementary Fig. 4 for results from assays controlling for amplicon size and quality). Increasing the ribonucleoprotein concentration three-fold did not improve detection in either case. These results reveal design principles for in vitro diagnostics, highlighting how primer design can be leveraged to obtain a suitable R-loop configuration and optimize CRISPR-Cas9-based nucleic acid detection.

Furthermore, we investigated the impact of perturbing the nuclease function. According to Chen et al9., RuvC is the domain responsible for trans-cleavage. Using the HNH-mutant Cas9 (working like a nickase)21 and crRNA-tracrRNAs, we noted that such a domain also plays a role in trans-cleavage (Fig. 5A; see also the null functionality of the RuvC-mutant Cas9 in Supplementary Fig. 5A, B)9. Absolute fluorescence levels associated with trans-cleavage were substantially lower, despite the targeting ability being roughly maintained (see insets of Fig. 5C). The effect of the HNH domain was further evidenced by using crRNAs of different lengths and relative units. While the wild-type nuclease was still able to process the poly(T) probe with significant efficacy when using a crRNA with a 22 nt spacer, the HNH-mutant Cas9 failed to do so (#1 vs. #1c or #2), only displaying collateral activity if guided by crRNAs with a 21 nt spacer at most (#1b or #4). Moreover, this mutant tolerated less well the use of sgRNAs (Fig. 5B). The time-dependent fluorescence signals associated with the cis- and trans-actions (for system #1) were also monitored (Fig. 5C), and a gel electrophoretic assay showed the absence of double breaks (Supplementary Fig. 5C). To broaden the study of the HNH domain effect, we focused on system #2 because of the absence of trans-cleavage activity, even when targeting an ssDNA molecule (Fig. 5A). Interestingly, employing a nicked dsDNA input in the targeted strand, we found that the HNH-mutant Cas9 displayed similar collateral activity to the wild-type nuclease (Fig. 5D). Arguably, a break in the targeted strand provides to Cas9 the required structural flexibility to efficiently activate the RuvC domain22. Nonetheless, guiding the mutant nuclease with a new crRNA in which the spacer was elongated from 22 nt to 31 nt, we did not observe the recovery of the signal (Supplementary Fig. 5D).

To gain mechanistic insight, we analyzed a set of three-dimensional structures23,24 deposited in the Protein Data Bank (PDB). A capping of the 5′ end of the sgRNA, with contacts with the RuvC domain, was observed in a structure (#5F9R) corresponding to a CRISPR-Cas9 complex bound to a short dsDNA molecule in which a strict 20 nt spacer follows, and the R-loop presents an unflanked configuration23 (Fig. 6A). This structure captures the catalytically competent, R-loop-forming state of Cas9, just before it cleaves the target. Such a capping, which does not occur when the spacer is elongated, might contribute to having an optimal configuration of the RuvC domain for an efficient trans-cleavage activity. Here, the very same dsDNA target and sgRNA from structure #5F9R were chemically synthesized to conduct a functional analysis. We demonstrated that a spacer elongation by 4 nt of that sgRNA led to a significant reduction in trans-cleavage activity (Fig. 6B). This reinforces the notion that the native 20 nt spacer leads to a fully functional S. pyogenes Cas9 in terms of catalysis. In addition, it is known that Cas9 adopts a more compact conformation around the central channel once it is primed for catalysis, especially because the HNH domain docks at the scissile phosphate of the targeted strand and the recognition lobe reorients to stabilize DNA binding23. The HNH domain appears to position even closer to RuvC domain when the R-loop is flanked in the RNA 5′ side as a result of targeting a long dsDNA molecule24,25, as structure #5Y36 shows (Fig. 6C). This structure also describes the catalytically competent state and involves an sgRNA with an elongated spacer (GG overhang in its 5′ end, which is exposed to the solvent). Here, as before, the very same dsDNA target and sgRNA from structure #5Y36 were chemically synthesized. We found that trans-cleavage activity significantly increased by trimming the dsDNA target in the RNA 5′ side to have an unflanked R-loop (Fig. 6D). A closer arrangement of the HNH and RuvC domains was also observed if the spacer has no overhang26 (Supplementary Fig. 6). In these conditions of flanked R-loop (natural scenario), the RuvC active site might become less solvent-accessible, leading to lower processing ability of outer ssDNA molecules. Yet, removal of the 5′ duplexed flank appear to loosen the complex, as the contacts with the positively charged cleft of the recognition lobe are lost25, hence altering the native conformation and potentially enhancing RuvC accessibility. This information provides, at least in part, a structural basis for the differential collateral activity here reported, stressing the high plasticity of the Cas proteins.

Conclusively, this work provides overlooked structural determinants of the CRISPR-Cas9 trans-cleavage activity. Flanked R-loops in the RNA 5′ side and elongated spacers beyond the canonical 20 nt are detrimental. Moreover, a catalytically active HNH domain favors trans-cleavage activity, despite it is accomplished by the RuvC domain, which aligns with the fact that these two nuclease domains act in concert22. It seems that S. pyogenes Cas9 evolved to prioritize precise on-target dsDNA cleavage, but these findings expand our view to consider as well its restricted collateral activity. We expect they will inspire developments in biological engineering and diagnostics. Further work is needed to define sequence determinants, resolve atomic structures in additional functional states, and explore trans-cleavage behavior across nuclease variants. As our understanding of CRISPR-Cas systems grows, so does our capacity to translate them into tools that address societal challenges2.

Methods
Molecular elements
Target DNA molecules were chemically synthesized and purified by IDT. They were labeled with the green fluorophore 6-Carboxyfluorescein (FAM) and the dark quencher Iowa Black FQ in their PAM-distal ends, so that we could monitor the targeting ability. The IDT OligoAnalyzer tool was used to calculate the melting temperature of the duplex flanking the R-loop in the RNA 5′ side of some target molecules, specifying a DNA concentration of 40 nM and a Mg2+ concentration of 12.5 mM. Poly(T) ssDNA probes of 18 nt and 13 nt, as well as a poly(A) ssDNA probe of 18 nt, were chemically synthesized and purified by IDT. They were labeled with the far-red fluorophore Cyanine5 (Cy5) and the dark quencher Iowa Black RQ in their ends, so that we could monitor the trans-cleavage activity. Three versions of S. pyogenes Cas9 (IDT) were used: the wild-type nuclease, the H840A nickase (HNH mutant), and the D10A nickase (RuvC mutant)21. In addition, the sgRNAs, crRNAs, and tracrRNA were produced in vitro with the TranscriptAid T7 high-yield transcription kit (Thermo) from DNA templates. They were purified using the RNA Clean and concentrator column (Zymo) and quantified in a spectrophotometer (NanoDrop, Thermo). The crRNA to target the E gene of SARS-CoV-2 with a canonical spacer of 20 nt was chemically synthesized (IDT). Sequences provided in Supplementary Data 1.

CRISPR-Cas9 reactions
Reactions were performed in 1x Tris/Acetate/EDTA (TAE) buffer pH 8.5 (Invitrogen), 0.05% Tween 20, and 12.5 mM MgCl2 at a final volume of 20 µL. 200 nM CRISPR-Cas9 ribonucleoprotein, previously assembled at room temperature for 30 min, was mixed with 40 nM target DNA (in the form of dsDNA, ssDNA, or plasmid) and 100 nM ssDNA probe. Control reactions lacking target DNA or CRISPR components were also considered. Reactions were incubated at 37°C for 1 h in tubes in a thermocycler (Mastercycler, Eppendorf). For time-dependent activity analyses, reactions were directly incubated at 37°C for 1 h in a microplate in the multimode plate reader.

Fluorescence quantification
CRISPR-Cas9 reaction volumes were loaded in a black 384-well microplate with clear bottom (Falcon), which was then placed in a multimode plate reader (CLARIOstar Plus, BMG) to measure green and far-red fluorescence. For FAM, excitation was at 490/8 nm and emission at 526/12 nm (green); for Cy5, excitation was at 640/8 nm and emission at 675/12 nm (far-red). The fluorescence values of the reaction buffer were subtracted to correct the signals. Absolute or relative fluorescence values were represented in the figures.

Nucleic acid detection assays
Samples of total RNA from human embryonic kidney (HEK293T) cells at 5 ng/μL, extracted using RNAzol RT (Merck), were spiked with the complete and purified RNA genome of SARS-CoV-2 (Vircell) at the final concentration of 5·103 copies/μL. A pre-amplification step by RT-RPA was performed with TwistAmp basic kit (TwistDX). For that, 500 U RevertAid (Thermo), 50 U RNase inhibitor (Thermo), and 480 nM custom forward and reverse primers (targeting the E gene) were added to 29.5 µL rehydration buffer. The TwistAmp basic reaction pellet was resuspended, and 2 µL spiked sample and 14 mM magnesium acetate were added to start the reaction. Reactions were incubated at 42°C for 30 min in a thermoblock (Thermomixer, Eppendorf). RT-RPA products were purified and quantified. Subsequently, CRISPR-Cas9 reactions were performed as previously described, and fluorescence readouts were obtained. In this case, the fluorescence value of the unprocessed probe was subtracted to correct the signals. Control CRISPR-Cas9 reactions were performed with RT-PCR products. Moreover, RT-RPA products were validated by quantitative PCR (qPCR). In MicroAmp fast reaction tubes with cap (Applied), 1 ng purified material, 500 nM primers, and Power SYBR Green PCR Master Mix (Applied) were combined for a total volume of 20 μL. Tubes were loaded into a qPCR system (QuantStudio 3, Applied) for fluorescence measurement, and the following protocol was employed: an initial activation step (95°C for 10 min) followed by 40 cycles of denaturation (95°C for 15 s) and annealing and extension (65°C for 1 min). Samples with CT > 30 were considered negative.

Gel electrophoresis
Nucleic acid cleavage by Cas9 was confirmed by agarose gel electrophoresis. For that, CRISPR-Cas9 reactions were first treated with 10 µg proteinase K (Ambion), incubating at 50°C for 1 h. The digestion was inactivated at 95°C for 15 min. Subsequently, 1 U RNase H (Ambion) and 2 µg RNase A (Invitrogen) were added, incubating at 37°C for 1 h. Samples were then loaded on a 1% or 3% agarose gel prepared with 0.5x Tris/Borate/EDTA (TBE) buffer, which was run for 45 min at room temperature (110 V). Gels were stained using GreenSafe (NZYtech). The GeneRuler DNA ladder mix (100–10000 bp, Thermo) and ultra-low range DNA ladder (10–300 bp, Thermo) were used as markers.

Structural models
Three different structures of a CRISPR-Cas9 complex bound to a target DNA molecule were analyzed: i) the crystal structure of a complex bound to a short dsDNA molecule in which the sgRNA has a strict 20 nt spacer (PDB #5F9R)23, ii) the cryogenic electron microscopy (cryo-EM) structure of a complex bound to a long DNA molecule in which the sgRNA has a 20 nt spacer with a three-nucleotide mismatch at positions 18–20 (PDB #7S4X)26, and iii) the cryo-EM structure of a complex bound to a long dsDNA molecule in which the sgRNA has a 22 nt spacer (GG overhang in the 5′ end; PDB #5Y36)24. These structures were visualized and aligned using PyMOL27. In the structures #5F9R and #7S4X, Cas9 was catalytically active; while in the structure #5Y36, Cas9 was catalytically dead (harboring the D10A and H840A mutations). All structures correspond to the D state, defined by the cleavage-ready positioning of the HNH domain.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have taken steps to address the reviewer’s comments, including the introduction of additional data as well as reducing the emphasis on the prior structure, and have moved the manuscript to Nature Communications. While many points have been sufficiently addressed, there are some lingering points that require more:\nThe authors still need to address how these insights could assist with molecular diagnostics. If anything, the requirement for a short end is limiting if pre-amplification is used. If post-amplification is used, then the starting DNA would need to be truncated somehow. This would represent an important limitation based on the authors’ insights that needs to be described in at least a few sentences.\nFigure 1: the tracrRNA should be noted as well, as a crRNA alone will not drive targeting by Cas9.\nL.66 and elsewhere: I recommend specifying cis-cleavage instead of targeting to more directly contrast with trans-cleavage. Targeting can still be used to describe when there is a match between the gRNA guide and the DNA.\nL.77 – 78: This DNA-free trans-cleavage was also observed with Cas12 nucleases that could be good to mention (PMID = 10.1038/s41467-024-50243-x), showing that this activity is not out of the question with Cas9.", "reviewer_2": "In this manuscript, the authors demonstrate that unflanked R-loop structures in the PAM-distal region facilitate trans-cleavage activity when Cas9 targets short double-stranded or single-stranded DNA substrates. They further show that extending guide RNA spacers beyond the canonical 20 nucleotides, even by only a few bases, markedly reduces this collateral cleavage activity. The authors provide evidence that catalytic activity in the HNH domain has a significant impact on cleavage efficiency. Finally, these findings are supported by structural analyses of relevant Protein Data Bank (PDB) entries. However, the following points should be addressed:\nMajor comments：\n1.The authors introduced five systems. However, the descriptions provided in the main text are vague and overly general, making it difficult for readers to find their differences. These systems are repeatedly mentioned throughout the Results and Discussion section; the differences among them can only be identified by referring to the supplementary figures. I recommend presenting these systems directly in the main figures to improve clarity and accessibility.\n2.In lines 101–106, the authors examined crRNAs with imperfect spacers for systems 1 and 2 and reported that a three-nucleotide mismatch occurred at positions 4, 5, and 6. I suggest that the authors provide detailed information on these nucleotides in Figure 2; otherwise, readers are unable to identify the location of the mismatch. Furthermore, the rationale for beginning with positions 4–6 and then moving to position 13 is unclear. What about the positions between 6 and 13? Can Cas9 tolerate single-nucleotide mismatches at these untested positions? In addition, the authors tested single-nucleotide mismatches at positions 5 and 13, as well as a three-nucleotide mismatch at positions 4–6. However, the effects of other possible variants, such as single mismatches at positions 4 and 6 or a two-nucleotide mismatch at positions 4 and 5, remain unexplored.\n3. In this manuscript, the authors reported that a flanked R-loop, an elongated spacer, and an inactive HNH domain can impede the trans-cleavage activity of Cas9. They further conclude that these findings support advances in nucleic acid detection. I recommend that the authors provide a real-world example demonstrating how their findings could improve the design of the CRISPR/Cas9 system for nucleic acid detection, thereby strengthening the practical relevance of their study.", "reviewer_3": "In this article, Montagud-Martínez, R. et al. investigate that the formation of flanked R-loops, an intervening sequence exceeding 20 nucleotides, and the inactivation of the HNH domain can all result in a decrease in the trans-cleavage activity of Cas9. The author investigated the trans-cleavage activity of Cas9 using a series of different target DNAs and analyzed the existing structure of flanked R-loop and an elongated spacer impeding trans-cleavage by Cas9. Despite rigorous experimentation, there is a lack of direct and clear evidence to support the arguments. Therefore, this work does not meet the standard for publication in Nature Communications.\n1. When the authors compared the cis- and trans-cleavage activities with system#1~5, there was basically no significant difference in the trans-cleavage activity of the system #3/4/5 regardless of the extension of the target sequence. But the trans-cleavage activity of Cas9 with system#5 is always worse, could the authors explain this discrepancy?\n2. In Figures 3 and 4, some experiments only increased the spacer sequence length of crRNA, while others also increased the sequence length of the target DNA. It is hoped that the authors can add schematic diagrams to make the distinction more obvious for readers' understanding.\n3. The author believes that HNH domain activity is an important prerequisite for the activation of Cas9's trans-cleavage activity. In previous studies by Chen et al., it was also proposed that the inactivation of the HNH domain reduced the trans-cleavage activity when use target dsDNA, which presents a certain degree of repetition of arguments. Furthermore, the use of nicked dsDNA targets to activate Cas9 mutants does not affect the trans-cleavage activity, which is somewhat different from the TS breakage caused by conformational changes in the HNH domain. Can this mechanism be explained more deeply from a structural perspective?\n4. The molecular diagnostic application based on trans-cleavage activity of Cas9 is a topic of concern. Has the author evaluated nucleic acid detection based on relevant research and provided a better detection method?\n5. The author analyzed the effect of R-loop and active HNH domains on trans cleavage activity based on existing structures, but it seems that there is still a lack of clear explanation.\n6. When the author used nicked dsDNA to investigate the effect of HNH domain on trans-cleavage activity, only system#2 was selected, and there was a lack of experiments on other systems. The author seems to lack some logic and coherence when selecting different target DNA for experiments."}, "author_response": "Authors’ response: For this revision, we have performed nucleic acid detection assays. In particular, we have detected the RNA genome of SARS-CoV-2 using the CRISPR-Cas9 system and a pre-amplification step of RT-RPA (see the new Fig. 4, here included for a rapid assessment). We prepared samples of total RNA from human embryonic kidney (HEK293T) cells spiked with the complete and purified RNA genome of SARS-CoV-2 (Vircell). Methods section has been upgraded accordingly. We have included a reference to the SARS-CoV-2 genome (ref. 19) and a reference to the RPA method (ref. 20). This text has been added to the manuscript: “To place our findings in an applied context, we conducted nucleic acid detection assays. To this end, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)19 was considered as the nucleic acid of interest (Fig. 4A). Using custom primers targeting the E gene, dsDNA amplicons containing the PAM sequence AGG were generated isothermally by reverse transcription recombinase polymerase amplification (RT-RPA)20, and then CRISPR-Cas9 reactions were run with a suitable crRNA-tracrRNA (20 nt spacer; Fig. 4B). Two different amplicons were generated, one short (105 bp in total, pre-PAM stretch of 28 bp) and another long (188 bp in total, pre-PAM stretch of 111 bp). We only obtained a differential fluorescence readout in the case of the short amplicon, which leads to an unflanked R-loop configuration after targeting favored by temperature (Fig. 4C). Increasing the ribonucleoprotein concentration 3-fold did not improve detection in either case. These results reveal design principles to further advance in the field of in vitro diagnostics.” For this revision, we decided to remove the reference to nucleic acid detection in the abstract. We feel more comfortable with this version, as we here intend to highlight basic constraints of Cas9 trans-cleavage activity.\nFigure 1: the tracrRNA should be noted as well, as a crRNA alone will not drive targeting by Cas9.\nAuthors’ response: We have included tracrRNA in the appropriate labels of the figures.\nL. 66 and elsewhere: I recommend specifying cis-cleavage instead of targeting to more directly contrast with trans-cleavage. Targeting can still be used to describe when there is a match between the gRNA guide and the DNA.\nAuthors’ response: We have specified at that point “cis-cleavage”, indicating as well that we measure the DNA targeting ability as a proxy. We used a fluorogenic DNA substrate to monitor this cis-activity, which is why we use the term DNA targeting ability, as it is more precise in what the data show. We also performed gel electrophoretic assays to confirm cis-cleavage.\nL. 77 – 78: This DNA-free trans-cleavage was also observed with Cas12 nucleases that could be good to mention (PMID = 10.1038/s41467-024-50243-x), showing that this activity is not out of the question with Cas9.\nAuthors’ response: We have included this reference in the paper.\nReviewer #2\nAuthors’ response: We have included schematics of the different systems analyzed in Fig. 1 (here included for a rapid assessment) to improve clarity of the paper. More details of them are provided in Fig. S1.\n2. In lines 101–106, the authors examined crRNAs with imperfect spacers for systems 1 and 2 and reported that a three-nucleotide mismatch occurred at positions 4, 5, and 6. I suggest that the authors provide detailed information on these nucleotides in Figure 2; otherwise, readers are unable to identify the location of the mismatch. Furthermore, the rationale for beginning with positions 4–6 and then moving to position 13 is unclear. What about the positions between 6 and 13? Can Cas9 tolerate single-nucleotide mismatches at these untested positions? In addition, the authors tested single-nucleotide mismatches at positions 5 and 13, as well as a three-nucleotide mismatch at positions 4–6. However, the effects of other possible variants, such as single mismatches at positions 4 and 6 or a two-nucleotide mismatch at positions 4 and 5, remain unexplored.\nAuthors’ response: We have expanded the analysis of fidelity with single-nucleotide mismatches at positions 8 and 19 and a two-nucleotide mismatch at position 5-6. We considered PAM-proximal positions (seed region) to mutate and also PAM-distal positions. We have also included the sequences of the crRNAs in the figure. We have added a reference to Kirillov et al., 2022 (ref. 14). This text has been added to the manuscript: “We assayed crRNAs with imperfect spacers for systems #1 and #2, introducing mismatches at various positions to probe R-loop formation and downstream cleavage activities. Single-nucleotide mismatches were introduced at positions 5, 8, 13, and 19. A two-nucleotide mismatch at positions 5, 6 and a three-nucleotide mismatch at positions 4, 5, 6 were considered too. Cas9 tolerated single-nucleotide mismatches across all positions with respect to cis-cleavage (Figs. 2A, 2C). Yet, collateral activity was more sensitive and sequence-dependent; it was substantially reduced by the mismatch at position 5 in system #1 and by the mismatch at position 13 in system #2 (Figs. 2B, 2D). Different basal activities were also noticed for each crRNA sequence. Furthermore, we observed that while the three-nucleotide mismatch impinged the formation of the complete R-loop in both systems, the two-nucleotide mismatch only did so in system #2 (Figs. 2A, 2C). These high-order mismatches nonetheless avoided a significant increase in trans-cleavage activity in both systems (Figs. 2B, 2D). These data underscore complexities associated with responsiveness and fidelity in the CRISPR-Cas9 system14.”\n3. In this manuscript, the authors reported that a flanked R-loop, an elongated spacer, and an inactive HNH domain can impede the trans-cleavage activity of Cas9. They further conclude that these findings support advances in nucleic acid detection. I recommend that the authors provide a real-world example demonstrating how their findings could improve the design of the CRISPR/Cas9 system for nucleic acid detection, thereby strengthening the practical relevance of their study.\nAuthors’ response: For this revision, we have performed nucleic acid detection assays. In particular, we have detected the RNA genome of SARS-CoV-2 using the CRISPR-Cas9 system and a pre-amplification step of RT-RPA (see the new Fig. 4, here included for a rapid assessment). We prepared samples of total RNA from human embryonic kidney (HEK293T) cells spiked with the complete and purified RNA genome of SARS-CoV-2 (Vircell). This text has been added to the manuscript: “To place our findings in an applied context, we conducted nucleic acid detection assays. To this end, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)19 was considered as the nucleic acid of interest (Fig. 4A). Using custom primers targeting the E gene, dsDNA amplicons containing the PAM sequence AGG were generated isothermally by reverse transcription recombinase polymerase amplification (RT-RPA)20, and then CRISPR-Cas9 reactions were run with a suitable crRNA-tracrRNA (20 nt spacer; Fig. 4B). Two different amplicons were generated, one short (105 bp in total, pre-PAM stretch of 28 bp) and another long (188 bp in total, pre-PAM stretch of 111 bp). We only obtained a differential fluorescence readout in the case of the short amplicon, which leads to an unflanked R-loop configuration after targeting favored by temperature (Fig. 4C). Increasing the ribonucleoprotein concentration 3-fold did not improve detection in either case. These results reveal design principles to further advance in the field of in vitro diagnostics.”\nReviewer #3\n1. When the authors compared the cis- and trans-cleavage activities with system#1~5, there was basically no significant difference in the trans-cleavage activity of the system #3/4/5 regardless of the extension of the target sequence. But the trans-cleavage activity of Cas9 with system#5 is always worse, could the authors explain this discrepancy?\nAuthors’ response: According to our data, trans-cleavage activity was significant in all systems analyzed and it was reduced when the target was extended (i.e., passing from an unflanked to a flanked R-loop). System #5 was indeed the one with the worst behavior, indicating sequence-dependent function. In this revised version, we have added a reference to Kirillov et al., 2022 (ref. 14) to acknowledge the “complexities associated with responsiveness and fidelity in the CRISPR-Cas9 system14.”\n2. In Figures 3 and 4, some experiments only increased the spacer sequence length of crRNA, while others also increased the sequence length of the target DNA. It is hoped that the authors can add schematic diagrams to make the distinction more obvious for readers' understanding.\nAuthors’ response: We have now schematics with added labels that indicate the variable regions.\n3. The author believes that HNH domain activity is an important prerequisite for the activation of Cas9's trans-cleavage activity. In previous studies by Chen et al., it was also proposed that the inactivation of the HNH domain reduced the trans-cleavage activity when use target dsDNA, which presents a certain degree of repetition of arguments. Furthermore, the use of nicked dsDNA targets to activate Cas9 mutants does not affect the trans-cleavage activity, which is somewhat different from the TS breakage caused by conformational changes in the HNH domain. Can this mechanism be explained more deeply from a structural perspective?\nAuthors’ response: We acknowledge that Chen et al. used the HNH-mutant Cas9 to analyze trans-cleavage activity (indeed, we have different citations to Chen et al. in our manuscript). At the beginning of the corresponding passage, we have changed “we here found that…” by “we noted that…”, adding ref. 9 at this point. In any case, we here incorporate to the analysis the effect of spacer length, which is critical even if it changes by a few bases. Moreover, our data shown in Fig. 5D (here included for a rapid assessment) do indicate that trans-cleavage activity is recovered upon using a nicked DNA in the case of system #2 with the HNH-mutant Cas9. This observation suggests that the presence of a nick —specifically in the targeted strand— facilitates a conformational state that mimics or bypasses the normal requirement for HNH domain-mediated cleavage to trigger the allosteric activation of the RuvC domain responsible for trans-cleavage (which is noticed here by using a crRNA of 22 nt spacer instead of 20 nt). We agree that the mechanism by which nicked DNA stimulates trans-cleavage activity in HNH-inactive Cas9 variants warrants a deeper structural investigation in future work.\n4. The molecular diagnostic application based on trans-cleavage activity of Cas9 is a topic of concern. Has the author evaluated nucleic acid detection based on relevant research and provided a better detection method?\nAuthors’ response: For this revision, we have performed nucleic acid detection assays. In particular, we have detected the RNA genome of SARS-CoV-2 using the CRISPR-Cas9 system and a pre-amplification step of RT-RPA (see the new Fig. 4, here included for a rapid assessment). We prepared samples of total RNA from human embryonic kidney (HEK293T) cells spiked with the complete and purified RNA genome of SARS-CoV-2 (Vircell). We would like to note that our goal with this work was to highlight constraints associated with Cas9 function, not to provide a better detection method. This text has been added to the manuscript: “To place our findings in an applied context, we conducted nucleic acid detection assays. To this end, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)19 was considered as the nucleic acid of interest (Fig. 4A). Using custom primers targeting the E gene, dsDNA amplicons containing the PAM sequence AGG were generated isothermally by reverse transcription recombinase polymerase amplification (RT-RPA)20, and then CRISPR-Cas9 reactions were run with a suitable crRNA-tracrRNA (20 nt spacer; Fig. 4B). Two different amplicons were generated, one short (105 bp in total, pre-PAM stretch of 28 bp) and another long (188 bp in total, pre-PAM stretch of 111 bp). We only obtained a differential fluorescence readout in the case of the short amplicon, which leads to an unflanked R-loop configuration after targeting favored by temperature (Fig. 4C). Increasing the ribonucleoprotein concentration 3-fold did not improve detection in either case. These results reveal design principles to further advance in the field of in vitro diagnostics.”\n5. The author analyzed the effect of R-loop and active HNH domains on trans cleavage activity based on existing structures, but it seems that there is still a lack of clear explanation.\nAuthors’ response: We analyzed PDB structure #5F9R regarding spacer length and PDB structure #5Y36 regarding R-loop configuration. To clarify our arguments regarding the effect of R-loop, we have added the following text: “Yet, removal of the 5’ duplexed flank appears to loosen the complex, as the contacts with the positively charged cleft of the recognition lobe are lost25, hence altering the native conformation and potentially enhancing RuvC accessibility.” We have also added the following sentence in that passage of the manuscript: “This reinforces the notion that the native 20 nt spacer leads to a fully functional S. pyogenes Cas9 in terms of catalysis.” Of course, we agree that further structural investigation is needed to provide deeper mechanistic insight on why CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop and an elongated spacer.\n6. When the author used nicked dsDNA to investigate the effect of HNH domain on trans-cleavage activity, only system#2 was selected, and there was a lack of experiments on other systems. The author seems to lack some logic and coherence when selecting different target DNA for experiments.\nAuthors’ response: We focused on system #2 for a further analysis because trans-cleavage activity was almost absent in this case with the HNH-mutant Cas9. This has been clarified in the text. Note that we performed experiments with HNH- and RuvC-mutant Cas9s for a variety of systems, not only for one. In addition, we have now performed an additional experiment with the HNH-mutant Cas9 to show that a crRNA with elongated spacer (31 nt) is not able to recover trans-cleavage activity when the target DNA is nicked (presented as the new Fig. S4D, here included for a rapid assessment), which further supports our conclusions. This text has been added to the manuscript: “To broaden the study of the HNH domain effect, we focused on system #2 because of the absence of trans-cleavage activity, even when targeting an ssDNA molecule (Fig. 5A). Interestingly, employing a nicked dsDNA input in the targeted strand, we found that the HNH-mutant Cas9 displayed similar collateral activity to the wild-type nuclease (Fig. 5D). Arguably, a break in the targeted strand provides to Cas9 the required structural flexibility to efficiently activate the RuvC domain22. Nonetheless, guiding the mutant nuclease with a new crRNA in which the spacer was elongated from 22 nt to 31 nt, we did not observe the recovery of the signal (Fig. S4D).”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have taken notable steps to improve the manuscript, including providing better illustrations to convey the tested target/guide configurations as well as including a demonstration of SARS-CoV-2 RNA detection tied to their findings. While the other concerns have been sufficiently addressed, the demonstration of RNA detection was insufficient. In particular, it was unclear how their results directly connect to the molecular insights from their work and whether the reported difference is due to merely using a smaller amplicon. More controls are needed, and there should be explicit statements tying these results to specific findings. The generated figure would also benefit from indicating where exactly Cas9 is targeting with accurate scaling.\nIn general, I recommend that the authors revisit their conclusions often appearing at the end of different paragraphs to ensure the conclusion better tie into the presented results without being too vague.", "reviewer_2": "The authors have taken steps to address our comments. I am satisfied with the revisions and have no further comments.", "reviewer_3": "The author has made revisions based on the reviewer's comments. However, there are still some unresolved issues.\n1. Line 108: For system # 2, the mutation occurring at position 13 has a significant impact on the trans-cleavage activity of Cas9. However, in Fig.S1A, the short dsDNA of system # 2 uses 21 nt, while the spacer of sgRNA is 22 nt. This elongated spacer length is shown to significantly inhibit its trans-cleavage activity in Fig.3D. Is there a possibility of reverse effects when conducting mutation site validation in this situation?\n2. The author found that the length of the spacer and the formation of the flanked R-loop both inhibit the trans-cutting activity of Cas9. The results in Fig.3A, 3B, and 3D show that extending the 5 'end PAM length of dsDNA to 23nt has no significant effect on its trans-cutting activity, while extending the spacer length to 21 nt begins to significantly decrease. In this case, the formation of a 23bp RNA-DNA double strand is even more inhibited. Does this indicate that the spacer length of RNA actually has a greater impact on its trans-cutting activity?\n3. The author's research has verified the effects of spacer length, formation of flanked R-loop, and HNH domain on the trans-cleavage activity of Cas9. This study has certain significance, but lacks some innovation. It is hoped that the author can further conduct relevant experiments in the future."}, "author_response": "Authors’ response: We have performed control experiments for the detection assays. On the one hand, we have confirmed that DNA amplicon size per se is not responsible for the differential readout obtained. Cas9 trans-cleavage was activated similarly from short and long PCR products provided the R-loop is unflanked in the RNA 5' side (see new results in Fig. S4C). On the other hand, the DNA products from the RT-RPA pre-amplification step were validated by qPCR, indicating that both primer sets (F1+R and F2+R) produce suitable material (see new results in Fig. S4D). In addition, we have enlarged the text to clarify how our results connect to the molecular insights reported.\nAuthors’ response: We have rewritten the conclusion sentence of the first paragraph of Results section as: “These results hence confirm the new catalytic feature and indicate that the magnitude of both cis- and trans-actions varies systematically with sequence composition.”\nWe have also rewritten the conclusion sentence of the fifth paragraph of Results section as: “Thus, our data demonstrate that spacer length is a critical and predominant variable to quantitatively control the collateral activity of Cas9, with progressive spacer elongation markedly suppressing trans-cleavage while preserving target binding and cis-cleavage competence.”\nWe have also rewritten the conclusion sentence of the sixth paragraph of Results section as: “These results reveal design principles for in vitro diagnostics, highlighting how primer design can be leveraged to obtain a suitable R-loop configuration and optimize CRISPR-Cas9-based nucleic acid detection.”\n--\nReviewer #3:\nAuthors’ response: In the case of system #2, the use of a crRNA with 22 nt spacer might puzzle the detrimental effect of the mismatch at position 13. Yet, the results obtained with crRNAs having mismatches at positions 5, 8, and 19 exclude this to be a general effect. To shed light, in this revision, we have conducted an additional experiment using a crRNA with 21 nt spacer and a mismatch at position 13, finding a small increase in trans-cleavage, although not a total recovery of the signal (see new results in Fig. S2F). This indicates that position 13 is critical in the case of system #2. Note that it is difficult to in vitro transcribe a crRNA with 20 nt spacer as the nucleotide at position 20 is C.\n2. The author found that the length of the spacer and the formation of the flanked R-loop both inhibit the trans-cutting activity of Cas9. The results in Fig.3A, 3B, and 3D show that extending the 5 'end PAM length of dsDNA to 23nt has no significant effect on its trans-cutting activity, while extending the spacer length to 21 nt begins to significantly decrease. In this case, the formation of a 23bp RNA-DNA double strand is even more inhibited. Does this indicate that the spacer length of RNA actually has a greater impact on its trans-cutting activity?\nAuthors’ response: Indeed, the effect of spacer length has a greater impact on trans-cleavage activity. We have rewritten the conclusion sentence of the fifth paragraph of Results section as: “our data demonstrate that spacer length is a critical and predominant variable to quantitatively control the collateral activity of Cas9”.\n3. The author's research has verified the effects of spacer length, formation of flanked R-loop, and HNH domain on the trans-cleavage activity of Cas9. This study has certain significance, but lacks some innovation. It is hoped that the author can further conduct relevant experiments in the future.\nAuthors’ response: We thank once again for the critical assessment, which served to improve our manuscript. Certainly, we plan to conduct further research on this Cas9 activity both from fundamental and engineering perspectives."}]

Decision letter