(A) Arrangement of GeoCas9 domains across the primary sequence. The cryo-EM structure of GeoCas9 in complex with gRNA (PDB: 8JTR) shows poor resolution of HNH. The GeoRec2 domain from PDB: 8JTR (gray) is overlaid with our X-ray structure of GeoRec2 (red, PDB: 9B72). (B) 1H15N TROSY HSQC NMR spectrum of GeoRec collected at 850 MHz. Overlays of this spectrum with resonances from spectra of GeoRec1 (black) and GeoRec2 (blue) demonstrate a structural similarity between the isolated subdomains and intact GeoRec.

(A, B) Sites of selected mutations within GeoRec2, K267 and R332, are highlighted as purple sticks directly facing the RNA and DNA modeled from NmeCas9 (PDB ID: 6JDV), allowing for prediction of the binding orientation within GeoCas9. NMR chemical shift perturbations caused by the K267E (C) or R332A (D) mutations are plotted for each residue of GeoRec. Gray bars denote sites of line broadening, the blue bar denotes an unassigned region of GeoRec corresponding to the native Rec1-Rec2 linker, and the red bar indicates the mutation site. The red dashed line indicates 1.5α above the 10% trimmed mean of the data. Chemical shift perturbations 1.5α above the 10% trimmed mean are mapped onto K267E (E) and R332A (F) GeoRec (red spheres). Resonances that have broadened beyond detection are mapped as yellow spheres and the mutation sites are indicated by a black sphere and green arrow.

(A) CPMG relaxation dispersion profiles of all residues with evidence of μs-ms motion, fit to a global kex of 147 ± 41 s-1 (WT GeoRec2, left), 376 ± 89 s-1 (K267E GeoRec2, center), and 142 ± 28 s-1 (R332A GeoRec2, right) collected at 25 °C and 600 MHz. Residues are colored in accordance with Table S1. Relaxation dispersion profiles for individual resonances are shown in Figures S8-S10. (B) Sites exhibiting CPMG relaxation dispersion in (A) are mapped to GeoRec as blue spheres. Adjacent domains within the cryo-EM structure of GeoCas9 are also shown. (C) Per-residue NMR order parameters of WT (black), K267E, and R332A (red, separate plots) GeoRec.

(A) NMR chemical shift perturbations caused by gRNA binding to WT, K267E, and R332A GeoRec. Gray bars denote sites of line broadening, and the blue bar denotes an unassigned region of GeoRec corresponding to the flexible Rec1-Rec2 linker. The red dashed line indicates 1.5α above the 10% trimmed mean of the data. (B) Representative NMR resonance shifts caused by titration of 39nt gRNA into WT GeoRec. (C) NMR titration of 39nt gRNA into K267E (top) and R332A (bottom) GeoRec. The left panel of each pair demonstrates that minimal change in NMR chemical shift or resonance intensity is apparent at gRNA concentrations mimicking the WT titration. The right panel of each pair depicts the titration over a three-fold wider concentration range of gRNA, where shifts and line broadening are visible. Representative resonances are colored by increasing gRNA concentration in the legend.

Representative MST-derived profiles of WT (A), K267E, and R332A (B) GeoRec binding to a Cy5-labeled 39nt gRNA, yielding Kd = 3.3 ± 1.5 µM, Kd = 7.2 ± 1.0 µM and Kd = 7.2 ± 1.5 µM, respectively. Bar graphs comparing Kd values across n ≥ 3 replicate samples are shown for Tnnt2 gRNA (C) and 8UZA gRNA from a recent cryo-EM structure (D). *p < 0.05, **p < 0.004

Effects of mutations in full-length GeoCas9 revealed by MD simulations. (A) The structure of GeoCas9 (PDB: 8UZA, protein in gray) bound to gRNA (orange) and DNA (magenta) is shown. Mutations studied include K267E (red), R332A (blue), and the 10 mutations of iGeoCas9 (lime green), all highlighted in surface representation. (B) Differential root-mean-square fluctuations (ΔRMSF) of protein residues computed between WT GeoCas9 and the K267E (red), R332A (blue), and double mutant (pink). (C) Distribution of protein-RNA contacts for WT and GeoCas9 variants computed over the 6 μs simulation ensemble. (D) Comparison of gRNA binding free energy to the Rec domain in WT GeoCas9 and variants. (E) Representative snapshots from MD simulations illustrating structural changes in Rec-gRNA association in WT GeoCas9 (left) and variants (right).

(A) Representative MST-derived profiles of WT, K267E, and R332A GeoCas9 binding to a Cy5-labeled full-length Tnnt2 gRNA. (B) Bar graph comparing Kd values across n ≥ 3 replicate samples are shown for Tnnt2 and 8UZA gRNA from a recent cryo-EM structure of GeoCas9. *p < 0.01