geocas9  (Addgene inc)

Journal: eLife

Article Title: Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

doi: 10.7554/elife.99275

Figure Lengend Snippet: Figure 1. Architecture of GeoCas9 and the GeoRec domain. (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.

Article Snippet: The full- length GeoCas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described (Harrington et al., 2017).

Techniques: Sequencing, Cryo-EM Sample Prep, Isolation

Journal: eLife

Article Title: Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

doi: 10.7554/elife.99275

Figure Lengend Snippet: Figure 2. Impact of single-point mutations on the GeoRec structure. (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 1.5σ above the 10% trimmed mean of the data. Chemical shift perturbations 1.5σ 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.

Article Snippet: The full- length GeoCas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described (Harrington et al., 2017).

Techniques: Binding Assay, Residue, Mutagenesis

Journal: eLife

Article Title: Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

doi: 10.7554/elife.99275

Figure Lengend Snippet: Figure 3. Single-point mutations enhance millisecond motions of GeoRec2. (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 Supplementary file 1. Relaxation dispersion profiles for individual resonances are shown in Figure 3—figure supplements 2–4. (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.

Article Snippet: The full- length GeoCas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described (Harrington et al., 2017).

Techniques: Dispersion, Cryo-EM Sample Prep, Residue

Journal: eLife

Article Title: Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

doi: 10.7554/elife.99275

Figure Lengend Snippet: Figure 6. 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 (∆RMSF) 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).

Article Snippet: The full- length GeoCas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described (Harrington et al., 2017).

Techniques: Mutagenesis, Comparison, Binding Assay

Journal: eLife

Article Title: Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

doi: 10.7554/elife.99275

Figure Lengend Snippet: Figure 7. Binding affinities determined for two guide RNAs to GeoCas9. (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 technical replicate samples are shown for Tnnt2 and 8UZA gRNA from a recent cryo-EM structure of GeoCas9. *p<0.01.

Article Snippet: The full- length GeoCas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described (Harrington et al., 2017).

Techniques: Binding Assay, Derivative Assay, Labeling, Cryo-EM Sample Prep

Journal: The Journal of chemical physics

Article Title: Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.

doi: 10.1063/5.0128815

Figure Lengend Snippet: FIG. 4. K597A is a control switch of thermostability in GeoCas9. (a) RNPs of wt- or K597A GeoCas9 and sgRNA were incubated at 37, 60, 75, or 85 ○C for 10 min, after which the RNPs were used for individual cleavage reactions at 37 ○C for 30 min. in vitro cleavage assays indicate that the K597A mutation does not effect on-target DNA cleavage at 37 and 60 ○C. K597A GeoCas9 is also able to cleave target DNA at 75 and 85 ○C more efficiently than wt. RNP concentration in each lane (left-to-right) is 100, 200, 300, 600, 900, and 0 nM at each temperature tested. Molecular weight markers on agarose gels (top-to-bottom) are 1000, 800, 600, 400, and 200 base pairs. (b) Temperature-dependent CD spectra reveal that a K597A GeoCas9 (i.e., full-length) mutant is more thermostable than wt-GeoCas9 by ∼15 ○C. (c) Correlations between temperature-dependent proton and nitrogen chemical shifts in 1H–15N TROSY HSQC spectra of wt- and K597A GeoHNH are shown for the nitrogen (right) and proton (left) dimensions. Temperature-dependent shifts outside 95% confidence boundaries are highlighted in red.

Article Snippet: Full-length K597A GeoCas9 purification The K597A mutation was introduced into a GeoCas9 plasmid available on Addgene (No. 87700). wt-GeoCas9 and the K597A variant used for CD spectroscopy and in vitro cleavage assays were purified as previously described.4

Techniques: Control, Incubation, In Vitro, Mutagenesis, Concentration Assay, Molecular Weight, Circular Dichroism