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WIREs Comput Mol Sci
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Establishing the allosteric mechanism in CRISPR‐Cas9

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Abstract Allostery is a fundamental property of proteins, which regulates biochemical information transfer between spatially distant sites. Here, we report on the critical role of molecular dynamics (MD) simulations in discovering the mechanism of allosteric communication within CRISPR‐Cas9, a leading genome editing machinery with enormous promises for medicine and biotechnology. MD revealed how allostery intervenes during at least three steps of the CRISPR‐Cas9 function: affecting DNA recognition, mediating the cleavage and interfering with the off‐target activity. An allosteric communication that activates concerted DNA cleavages was found to led through the L1/L2 loops, which connect the HNH and RuvC catalytic domains. The identification of these “allosteric transducers” inspired the development of novel variants of the Cas9 protein with improved specificity, opening a new avenue for controlling the CRISPR‐Cas9 activity. Discussed studies also highlight the critical role of the recognition lobe in the conformational activation of the catalytic HNH domain. Specifically, the REC3 region was found to modulate the dynamics of HNH by sensing the formation of the RNA:DNA hybrid. The role of REC3 was revealed to be particularly relevant in the presence of DNA mismatches. Indeed, interference of REC3 with the RNA:DNA hybrid containing mismatched pairs at specific positions resulted in locking HNH in an inactive “conformational checkpoint” conformation, thereby hampering off‐target cleavages. Overall, MD simulations established the fundamental mechanisms underlying the allosterism of CRISPR‐Cas9, aiding engineering strategies to develop new CRISPR‐Cas9 variants for improved genome editing. This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics
Schematic representation of the CRISPR‐Cas adaptive immunity. (a) Upon viral infection, parts of invading nucleic acids are internalized into the CRISPR genetic array that subsequently transcribes and matures into a guide RNA complex containing a CRISPR RNA (crRNA) and trans‐CRISPR RNA (tracr‐RNA). (1) The Cas9 protein binds the guide RNA and uses its sequence to recognize complementary DNA sequences. Site‐specific recognition of the viral DNA is preceded by the binding of a short Protospacer Adjacent Motif (PAM), which enables the selection across the genome. (b) Three‐dimensional structure of the Streptococcus pyogenes CRISPR‐Cas9 (SpCas9) complex with a guide RNA and DNA (PDB code: 4UN3).10 Cas9 is shown in molecular surface, highlighting individual domains in different colors. The RNA (yellow), target DNA (TS, cyan), and nontarget DNA (NTS, violet) are shown as ribbons
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Conformational basis of off‐target effects. (a) Crystal structure of CRISPR‐Cas9 in a “conformational checkpoint” state (PDB code: 4UN3).10 The arrow indicates the conformational change required by the inactive HNH to cleave the DNA target strand (TS). (b) Extended opening of the RNA:DNA hybrid and newly formed interactions with the L2 loop (magenta), observed during MD simulations of CRISPR‐Cas9 in the presence of four base pair mismatches at PAM distal sites. The 692–700 α‐helix of the Rec3 region (black) is shown to insert within the within the RNA:DNA, promoting its extended opening. (c) RNA:DNA minor groove width computed along MD simulations of CRISPR‐Cas9 bound to an on‐target DNA (black) and in the presence of one to four PAM distal mismatches (left panel). A vertical bar indicates the experimental minor groove width (i.e., 11 Å from x‐ray crystallography). The minor groove width has been measured at the level of base pair 17 (right panel).19 Reprinted with permission from Reference 19 Copyright 2019 American Chemical Society. https://pubs.acs.org/doi/full/10.1021/acscentsci.9b00020. Further permissions related to the material excerpted should be directed to the American Chemical Society
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Interdependent dynamics of HNH and the REC2‐3 regions. (a) Matrices of the generalized correlations (GC, upper triangle) and of the per‐domain correlation score (Csi) (bottom triangle) computed for the activated state of CRISPR‐Cas9 identified by MD. The strength of the correlated motions is color coded green (highly correlated motions) to gray (not correlated). (b) The highest per‐domain coupled motions, involving HNH and the Rec2‐3 regions, are reported using double‐headed arrows.17 Reprinted with permission from Reference 17 Copyright 2018 Cambridge University Press. https://doi.org/10.1017/S0033583518000070
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HNH domain activation observed through long time scale MD simulations. (a) The HNH domain (green) is shown to approach the catalytic site on the DNA target strand (TS), changing conformation from a preactive X‐ray structure (PDB code: 5F9R) to the active state identified by MD (top panel). The time evolution of the distance between the catalytic H840 and the scissile phosphate (H840–PDNA) is computed along ~400 ns Gaussian accelerated MD (GaMD, central panel) and along ~16 μs of a continuous MD simulation using Anton2 (bottom panel). A black dashed line indicates the preactive conformation (PDB code: 5F9R), which has been used as a starting point for MD simulations. (b) Conformational change of REC2 and REC3 during the HNH activation, identified by single molecule FRET (smFRET) experiments (top panel). The evolution along a ~16 μs MD run of the smFRET pairs D273–E60 (middle panel) and S701–S960 (bottom panel). Transparent bars (pink) indicate the experimental distribution of the smFRET distances in the activated conformation.17 Reprinted with permission from Reference 17 Copyright 2018 Cambridge University Press. https://doi.org/10.1017/S0033583518000070
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Conformational change of HNH in the presence of the DNA nontarget strand (NTS). (a) Schematic representation of the HNH conformational transition toward the DNA target strand (TS, cyan) in the presence of the NTS (violet). The HNH domain (green) is shown to establish a number of interactions with the DNA NTS through the L2 loop (blue). (b) Time evolution of the distance between the catalytic H840 and the phosphorus atom of the scissile phosphate (PDNA), along MD simulations of CRISPR‐Cas9 bound to the NTS (i.e., with NTS) and without the NTS (i.e., w/o NTS).39 Reprinted with permission from Reference 39 Copyright 2016 American Chemical Society. https://pubs.acs.org/doi/full/10.1021/acscentsci.6b00218. Further permissions related to the material excerpted should be directed to the American Chemical Society
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PAM‐induced allostery in CRISPR‐Cas9. (a) Conformational space adopted by the CRISPR‐Cas9 complex spanning over the two principal components of motions (i.e., PC1 vs. PC2), computed over eight independent MD runs of Cas9 bound to PAM (i.e., wPAM) and without PAM (i.e., w/oPAM). (b) “Open‐to‐close” conformational transition identified along the first principal component. (c) Per‐domain correlation score (Csi) matrix, identifying the Cas9 inter‐domain coupled motions color‐coded green (correlated) to white (not correlated). (d) Community network graph of Cas9–w/oPAM (right) and wPAM (left). Bonds connecting communities correspond to the interconnection strength. (e) Allosteric transducer loops L1/L2 connecting the catalytic HNH and RuvC domain. Critical network nodes (Q771, E584, K775, and R905) of the L1/L2 loops forming essential edges in the allosteric transmission between HNH and RuvC.15 Reprinted with permission from Reference 15 Copyright 2017 American Chemical Society. https://pubs.acs.org/doi/10.1021/jacs.7b05313. Further permissions related to the material excerpted should be directed to the American Chemical Society
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