(A) Architecture of GeoCas9 modeled with AlphaFold2. The GeoRec2 domain from the model (grey) is overlaid with an X-ray structure of GeoRec2 (red, PDB ID: 9B72, RMSD 1.03 Å). (B) 1H-15N 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 subdomain and intact GeoRec. (C) NMR titration of a 39-nt gRNA into WT GeoRec. Representative resonances are colored by increasing gRNA concentration in the legend. (D) NMR chemical shift perturbations caused by gRNA binding to WT GeoRec. Gray bars denote sites of line broadening and the blue bar denotes an unassigned region of GeoRec corresponding to the native Rec1-Rec2 linker. The red dashed line indicates 1.5α above the 10% trimmed mean of the data. (E) Chemical shift perturbations >1.5α are mapped onto GeoRec (red spheres). Resonances that have broadened beyond detection are mapped as yellow spheres. (F) MST-derived binding affinity of GeoRec to the Cy5-labeled 39-nt gRNA yields a Kd = 2.95 ± 0.53 μM.

(A, B) Sites of selected mutations within GeoRec2, K267 and R332, are highlighted as purple sticks directly facing the nucleic acids 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 1.50 above the 10% trimmed mean of all shifts. Chemical shift perturbations >1.50 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 for all sites of μs-ms flexibility, fit to a global kex of 147 ± 41 s- 1 (WT GeoRec2, top), 376 ± 89 s-1 (K267E GeoRec2, middle), and 142 ± 28 s-1 (R332A GeoRec2, bottom). Relaxation dispersion profiles are colored according to Table S1. B) CPMG relaxation dispersion profiles for representative residues in GeoRec2. A mixture of curved and flat profiles illustrate the changes in and redistribution of μs-ms motions between WT and GeoRec2 variants. (C) All sites exhibiting CPMG relaxation dispersion plotted in A are mapped to GeoRec as blue spheres. Adjacent domains within the AlphaFold2 model of GeoCas9 are also shown.

(A) NMR titration of a 39-nt gRNA into K267E (top) and R332A (bottom) GeoRec. The left panel of each pair demonstrates that gRNA concentrations mimicking the WT titration induce minimal change in NMR chemical shift or resonance intensity. The right panel of each pair depicts the titration over a three-fold greater concentration range of gRNA, where shifts and line broadening are visible. Representative resonances are colored by increasing gRNA concentration according to the legend. (B) NMR chemical shift perturbations caused by gRNA binding to K267E and R332A GeoRec. Gray bars denote sites of line broadening and the blue bar denotes an unassigned region of GeoRec corresponding to the native Rec1-Rec2 linker. The red dashed line indicates 1.50 above the 10% trimmed mean of all shifts. (C) Chemical shift perturbations >1.50 are mapped onto GeoRec (red spheres). Resonances that have broadened beyond detection are mapped as yellow spheres. (D) Fitted thermal denaturation profiles derived from CD spectra of apo WT (black line), K267E (dashed line), and R332A (dotted line) GeoCas9 are shown on the left. Fitted denaturation profiles of the same proteins, as an RNP in complex with 141-nt gRNA, are shown in the right panel. The red dashed line denotes the Tm of the apo proteins, which are nearly identical. (E) MST-derived binding affinities of K267E and R332A GeoRec and a Cy5-labeled 39-nt gRNA yielding Kd = 6.67 ± 2.0 µM and Kd = 4.02 ± 1.34 µM, respectively.