Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9
- Department of Bioengineering and Department of Chemistry, University of California, Riverside, United States
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, United States
- International Centre for Theoretical Physics, Italy
- Department of Chemistry, Yale University, United States
Figures
Figure 1 with 2 supplements
Architecture of the Cas9 endonuclease (center, PDB: 5F9R) (Jiang et al., 2016) highlighting its protein domains as follows: HNH (green), RuvC (light blue), PAM-interacting (PI, orange), and recognition lobe (REC, gray).
A portion of the RNA:DNA hybrid behind HNH has been removed for clarity. Close-up view (left): the previously defined allosteric pathway spanning the HNH domain is shown (pink) and the locations of …
Figure 1—figure supplement 1
Structure of the HNH domain.
(A) The structure of the HNH domain (residues 775–908) crystallized within the full-length Cas9 (PDB: 5F9R, green) (Jiang and Doudna, 2017) is superposed on the X-ray structure of the HNH construct …
Figure 1—figure supplement 2
Circular dichroism of HNH mutants.
(A) The CD spectrum from 195 to 260 nm of wild-type (WT) HNH (black), K855A (red), K810A (blue), and K848A (green) showing that all three mutants are similar to the WT spectrum with significant …
Figure 2 with 1 supplement
Environmental perturbations (EP) caused by the K855A, K810A, and K848A mutations in HNH.
(A) The NMR composite chemical shifts (black bars) determined by are reported for each mutant. Blue lines are the 10% trimmed mean of all shifts and red lines represent 1.5σ above the 10% trimmed …
Figure 2—figure supplement 1
Environmental perturbations (EP) caused by the K855A, K810A, and K848A mutations in HNH.
The EP scores have been computed for the HNH domain (residues 775–908) in the full-length CRISPR-Cas9. Data are reported for three simulation replicas for each system. The site of mutation is …
Figure 3 with 4 supplements
Dynamic properties of HNH and its mutants.
(A) Structure of the HNH domain showing μs-ms timescale dynamics preserved following three K–to–A mutations (central panel). Spheres represent sites of Carr-Purcell-Meiboom-Gill (CPMG) relaxation …
Figure 3—figure supplement 1
Dynamic properties of the HNH mutants.
(A) Sites of ms timescale dynamics (magenta spheres) observed through Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (Loria et al., 1999) studies of the K855A, K810A, and K848A mutants are …
Figure 3—figure supplement 2
Per-residue Rex determined from Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments for wild-type (WT) (black), K855A (red), K848A (green), and K810A (blue) HNH.
Lower panels denote the Rex differences (ΔRex) for each K-to-A mutant from WT HNH (left) and the mutants against each other (right). The average Rex is shown for the mutant comparisons, …
Figure 3—figure supplement 3
Distribution of kex values determined from Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion for wild-type (WT) HNH and specificity-enhancing mutants.
K-to-A mutants skew the kex distribution toward slower values, with bin sizing determined with a protocol described by Scott in Biometrika, 1979, 66, 605. Data for WT HNH was previously reported in J…
Figure 3—figure supplement 4
Order parameters (S2) derived from model-free analysis of T1, T2, and 1H-[15N] nuclear overhouser effect (NOE) experiments.
Panels on the left show the per-residue S2 for K-to-A mutants (red) compared to wild-type (WT) HNH (black). Panels on the right show the change in order parameter (ΔS2 ) for each K-to-A mutant …
Figure 4 with 1 supplement
Alterations of the allosteric signaling in full-length CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) systems.
(A) Close-up view of the HNH domain within the wild-type (WT) full-length Cas9, showing seven communities of synchronized dynamics, indicated using different colors. Three communities are allosteric …
Figure 4—figure supplement 1
Circular graphs reporting the mutation-induced edge betweenness change (ΔEB) for the K855A, K810A, and K848A mutants in the HNH domain in the full-length Cas9 (A) and in its isolated form (B).
Data are reported upon averaging three replicas (see Appendix 1—figures 6 and 7). In these circular graphs, the HNH communities are disposed in a circle and are connected by links, whose thickness …
Figure 5 with 1 supplement
Residues exchanging between dynamic communities of the wild-type (WT) HNH domain and its mutants in full-length CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein).
The HNH communities identified through computational analysis are used as a reference, while the dynamic exchange among them is based on the number of residues displaying Carr-Purcell-Meiboom-Gill …
Figure 5—figure supplement 1
Residues exchanging between dynamic communities of the wild-type (WT) HNH domain and its mutants in the full-length CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) system (A) and in the isolated HNH domain (B).
The HNH communities identified through computational analysis are used as a reference, while the dynamic exchange among them is based on the number of residues displaying Carr-Purcell-Meiboom-Gill …
Figure 6
Critical allosteric hotspots of signal transmission through the HNH domain of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein).
(A) Circular graph showing that allosteric sites of HNH (A1–A3) display a loss in communication (red bond) upon alanine mutation of K855, K810, and K848, while the non-allosteric sites (NA1–NA4) …
Figure 7
Flexibility of the DNA non-target strand.
(A) Overview of an enlarged model system of the ild-type (WT) CRISPR-Cas9, including a longer DNA non-target strand reaching the K1003 and R1060 residues (PDB: 5Y36) (Jiang and Doudna, 2017). Cas9 …
Appendix 1—figure 1
Allosteric dynamical pathways across the HNH domain for the K855A, K810A, K848A mutants, computed from μs-length molecular dynamics (MD) of the full-length Cas9 (data are shown for three simulation replicas).
The residue-to-residue dynamical pathways optimizing the momentum transport from REC to RuvC are shown using orange spheres. The HNH domain (green) is shown on side view. The wild-type (WT) …
Appendix 1—figure 2
Allosteric dynamical pathways across the HNH domain for the K855A, K810A, K848A mutants, computed from μs-length molecular dynamics (MD) of the isolated HNH domain (data are shown for three simulation replicas).
The residue-to-residue dynamical pathways optimizing the momentum transport from REC to RuvC are shown using yellow spheres. The HNH domain (green) is shown on side view. The wild-type (WT) …
Appendix 1—figure 3
Comparison of Lange and Grubmüller’s generalized correlations (GCs) and Pearson’s correlations (PCs) for allosteric pathways analysis.
Difference matrices between the GCs and PCs (ΔGC–PC), computed for the wild-type (WT) HNH and the K855A, K810A, and K848A mutants in the full-length Cas9 (A) and in the isolated HNH domain (B). The …
Appendix 1—figure 4
Allosteric pathways derived from Pearson’s coefficients (PCs), computed for the wild-type (WT) HNH and the K855A, K810A, and K848A mutants in the full-length Cas9 (A) and in the isolated HNH domain (B).
Residues composing the HNH allosteric pathways are shown as orange spheres. The WT allosteric pathway previously identified through molecular dynamics MD and NMR (pink ribbons) (East et al., 2020a) …
Appendix 1—figure 5
Close-up view of the HNH domain within the wild-type (WT) CRISPR-Cas9 (A) and in its isolated form (B).
Seven communities of synchronized dynamics are shown using different colors. Data are reported for three simulation replicas. Three communities are allosteric (A1 yellow, A2 cyan, and A3 purple), …
Appendix 1—figure 6
(A–B) Two-by-two matrices of the edge betweennesses (EB), computed for the wild-type (WT) system and for the K855A, K810A, and K848A mutants.
The EB are computed for each couple of communities of HNH in the full-length Cas9 and are plotted according to the scale on the right. Data are reported for three simulation replicas (A), and for …
Appendix 1—figure 7
(A–B) Two-by-two matrices of the edge betweennesses (EB), computed for the wild-type (WT) system and for the K855A, K810A, and K848A mutants.
The EB are computed for each couple of communities of HNH in its isolated form and are plotted according to the scale on the right. Data are reported for three simulation replicas (A), and for the …
Appendix 1—figure 8
Community network plots (top) and corresponding two-by-two matrices (bottom) of the edge betweennesses (EB) computed for the wild-type (WT) HNH and for the K855A, K810A, and K848A mutants in the full-length Cas9 (A) and in the isolated HNH domain (B).
Bonds connecting communities are a representation of their EB and hence of the communities’ intercommunication strength. Data are reported for the average EB matrices in Appendix 1—figures 6 and 7. …
Additional files
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Supplementary file 1
Additonal MD simulationa dn NMR data pertaining to the allosteric role of specificity enhancement mutations in HNH.
- https://cdn.elifesciences.org/articles/73601/elife-73601-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/73601/elife-73601-transrepform1-v2.docx
