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  1. Author Response:

    Reviewer #2 (Public Review):

    The CRISP-Cas9 complex has revolutionized genomic editing techniques. The widespread application of this new molecular tool enables a precise and accurate DNA cleavage that has been impossible to achieve. Yet, in some cases, the system suffers from a lack of specificity. In this paper, the authors present a new study on the characterization of the allosteric communication within the CRISP-Cas9 complex. They identified three different mutations that disrupt the complex's internal allosteric communication, affecting the cleavage reaction's specificity to different extents. The authors argue that the various degrees of perturbation are correlated with the Cas9 specificity. Given the size of the complex, the authors utilize a divide and conquer approach to studying the structural-dynamic changes of the isolated HNH endonuclease using NMR spectroscopy. Then they used molecular dynamics simulations to relate the changes in the isolated enzyme to the entire complex. As marked by the authors, the effects of the selected mutations (K855A, K810A, and K848A) are minimal. The HSQC spectrum in Figure 2B shows only marginal chemical shift changes in the protein fingerprint. The latter is supported by the CD spectra that show no significant perturbations in the dichroic profiles. However, the lineshapes reveal substantial changes in the enzyme dynamics apparent from the broadening of several signals. The chemical shift perturbations, although small, show that K855A has the most pronounced spectroscopic changes followed by K810A and K848A. As expected, the most significant differences are revealed by relaxation studies. The authors performed T1, T2, and heteronuclear NOE experiments to characterize the fast dynamics of the protein in the NMR time scale, revealing the most significant differences in the K855A mutant.

    Additionally, they used CPMG dispersion experiments to analyze the dynamics in the micro-to-millisecond time scale. From these measurements, the authors conclude that the relaxation characteristics of the mutants do not change significantly, i.e., the mutants possess conformational flexibility similar to the wild type. To interpret the dynamic behaviors of the different HNH variants, the authors performed MD simulations and analyzed the allosteric network using community analysis. The computational work revealed the connections between the communities and how the mutants affect interdomain communication (figure 5).

    Overall the paper is exciting and shows how NMR and MD simulations can be used synergistically to dissect the intra- and inter-molecular allosteric communication in highly complex systems. However, there are a few shortcomings that the authors need to address. One significant concern is the lack of a direct comparison between the NMR studies and the MD simulations. Additionally, it is unclear how these dynamics or structural perturbations caused by these selected mutants are converted into the enzyme's increased or decreased specificity.

    Other technical concerns:

    A) The authors performed relaxation measurements for fast dynamics. However, they did not calculate the order parameters for the protein backbone. Usually, the order parameters from the protein backbone can be directly compared to the calculated values from MD trajectories. How do the S2 values from the two techniques compare?

    The reviewer is absolutely correct and we have now included S^2 parameters for each K-to-A mutant and determined the difference from WT HNH (new Figure Supplement S7). We also added discussion of these data on page 9, lines 185-189. Briefly, S^2 parameters for each mutant are quite similar to those of WT HNH, evidenced by DeltaS^2 values (greater or equal to) 0.1 for the majority of residues. These data also mirror DeltaS^2 values determined from MD simulations. Further, we note agreement between S^2 and the 1^H-[15^N] NOE that show depressed values sporadically between residues 800-825, surrounding residue 850, and at the C-terminus.

    B) The authors state that the differences in the relaxation dispersion profiles are less than 1.5 Hz, indicating small changes in dynamics. Did the author compare all residues or a subset of residues?

    C) In the discussion, the authors refer to the synchronous motions that may be responsible for specificity. How did they deduce that the motions are synchronous? From MD simulations or the global fitting of the CPMG curves? Do motions need to be synchronous for effective allosteric communications?

    In the manuscript, we referred to “synchronous” when describing community network analysis (CNA), where groups of residues displaying highly synchronized dynamics are gathered in communities. This wording is indeed employed in several computational studies harnessing CNA (PNAS, 2017, 114, E3414-E3423). We therefore did not employ the word “synchronous” as mechanistically. We have now changed our phrasing in the manuscript to avoid any confusion.

    D) Finally, the authors claim that mutations can target sites identified in this study (hotspots) to improve CRISP-Cas9 function. Can the authors elaborate more on this point? How do they envision mutations to tune the function of the complex?

    We thank the reviewers for this insightful comment, which gives us the opportunity to suggest critical hotspots for mutational studies. Our computational analysis indicated that the three K–to–A mutations mainly disrupt the cross-talk between the A1 and A2 communities (Figure 4). This effect is observed for all mutants, and is confirmed by the analysis of the NMR relaxation data (Figure 5), suggesting that the A1-A2 communities are critical hotspots for the signal transmission. Building on this observation, mutational studies targeting residues of the A1-A2 communities could impact the allosteric communication and, in turn, modulate the function and specificity of the system. We have now included this discussion in the main text (page 16, lines 332-341 and page 18, lines 393-395) adding Figure 6. The Abstract was also amended including this information.

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  2. Evaluation Summary:

    This is a comprehensive study combining solution NMR with molecular dynamics simulations to uncover the effects of three key mutations in the Cas9 HNH domain that increase CRISP-Cas9 complex specificity and reduce off-target activity. Through the analysis of these three different mutations, the authors concluded that by tuning the conformational dynamics of the HNH module in the CRISP-Cas9 complex, it is possible to control the function and specificity of the system. Combined these findings could have important implications for the design of new variants for this important gene editing complex.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Originally identified as a component of the bacterial adaptive immune response, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have found widespread biotechnology applications in genome editing, the detection and imaging of nucleic acids, gene silencing, and chromatin engineering. The focus of this paper is on the CRISPR-Cas9 system, which is paradigmatic for this class of protein complexes. Thus, understanding allosteric communication in CRISPR-Cas9 in relation to its ability to recognize and excise targeted nucleic acid segments is a topic of great current interest. Specifically, this study combines solution NMR with molecular dynamics simulations and employs graph-theoretical analysis of the protein network to uncover the effects of three key mutations in the HNH domain (K810A, K848A, and K855A) that increase Cas9 specificity and reduce off-target activity. These mutations were found to disrupt the main communication pathway between the RuvC and REC modules, suggesting a direct link between changes in the allosteric network and the observed increase in Cas9 target specificity. Notably, the magnitude of decrease in allosteric communication (as measured by mutation-induced edge betweenness difference) correlates perfectly to the order of specificity enhancement (K855A > K848A ~ K810A). Thus, the paper sheds light on an aspect of Cas9 function that may have important implications for the design of new gene editing tools with improved specificity. The findings are novel and well supported by the data. In particular, the molecular dynamics simulations and analysis appear to have been done using appropriate simulation protocols. Both the experimental and computational methods are described in detail. Approaching allosteric effects in Cas9 from multiple angles, using multiple experimental and computational techniques is a notable strength of this manuscript.

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  4. Reviewer #2 (Public Review):

    The CRISP-Cas9 complex has revolutionized genomic editing techniques. The widespread application of this new molecular tool enables a precise and accurate DNA cleavage that has been impossible to achieve. Yet, in some cases, the system suffers from a lack of specificity. In this paper, the authors present a new study on the characterization of the allosteric communication within the CRISP-Cas9 complex. They identified three different mutations that disrupt the complex's internal allosteric communication, affecting the cleavage reaction's specificity to different extents. The authors argue that the various degrees of perturbation are correlated with the Cas9 specificity. Given the size of the complex, the authors utilize a divide and conquer approach to studying the structural-dynamic changes of the isolated HNH endonuclease using NMR spectroscopy. Then they used molecular dynamics simulations to relate the changes in the isolated enzyme to the entire complex. As marked by the authors, the effects of the selected mutations (K855A, K810A, and K848A) are minimal. The HSQC spectrum in Figure 2B shows only marginal chemical shift changes in the protein fingerprint. The latter is supported by the CD spectra that show no significant perturbations in the dichroic profiles. However, the lineshapes reveal substantial changes in the enzyme dynamics apparent from the broadening of several signals. The chemical shift perturbations, although small, show that K855A has the most pronounced spectroscopic changes followed by K810A and K848A. As expected, the most significant differences are revealed by relaxation studies. The authors performed T1, T2, and heteronuclear NOE experiments to characterize the fast dynamics of the protein in the NMR time scale, revealing the most significant differences in the K855A mutant.

    Additionally, they used CPMG dispersion experiments to analyze the dynamics in the micro-to-millisecond time scale. From these measurements, the authors conclude that the relaxation characteristics of the mutants do not change significantly, i.e., the mutants possess conformational flexibility similar to the wild type. To interpret the dynamic behaviors of the different HNH variants, the authors performed MD simulations and analyzed the allosteric network using community analysis. The computational work revealed the connections between the communities and how the mutants affect interdomain communication (figure 5).

    Overall the paper is exciting and shows how NMR and MD simulations can be used synergistically to dissect the intra- and inter-molecular allosteric communication in highly complex systems. However, there are a few shortcomings that the authors need to address. One significant concern is the lack of a direct comparison between the NMR studies and the MD simulations. Additionally, it is unclear how these dynamics or structural perturbations caused by these selected mutants are converted into the enzyme's increased or decreased specificity.

    Other technical concerns:

    A) The authors performed relaxation measurements for fast dynamics. However, they did not calculate the order parameters for the protein backbone. Usually, the order parameters from the protein backbone can be directly compared to the calculated values from MD trajectories. How do the S2 values from the two techniques compare?

    B) The authors state that the differences in the relaxation dispersion profiles are less than 1.5 Hz, indicating small changes in dynamics. Did the author compare all residues or a subset of residues?

    C) In the discussion, the authors refer to the synchronous motions that may be responsible for specificity. How did they deduce that the motions are synchronous? From MD simulations or the global fitting of the CPMG curves? Do motions need to be synchronous for effective allosteric communications?

    D) Finally, the authors claim that mutations can target sites identified in this study (hotspots) to improve CRISP-Cas9 function. Can the authors elaborate more on this point? How do they envision mutations to tune the function of the complex?

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  5. Reviewer #3 (Public Review):

    In the associated manuscript, the authors aim to combine solution state NMR and MD simulations to characterize the structures and dynamics of three mutations within the HNH domain of the CRISPR-Cas9 system. These previously described mutants increase the specificity of the endonuclease through a proposed mechanism of altering the allosteric signaling of the enzyme. A precise understanding of this mechanism could allow for the production of next generation CRISPR-Cas9 systems with increased specificity.

    The authors use a 3.6 us MD simulation and interesting analysis methods to characterize the changes in structure and dynamics of the three mutants. Unfortunately, the NMR data plays a very minor supporting role and is not fully analyzed or described. Thus, many of the conclusions from these results are weakened, and it is unclear whether the results support their conclusion.

    If this work is predictive, it could be very interesting for the field. Off-target effects are a problem with gene editing via the CRISPR-Cas9 system, and this study could suggest novel mutants which may help further increase specificity. That promise remains to be seen.

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