pet-26-mbp-elic  (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: ( A ) Arrangement of Geo Cas9 domains across the primary sequence. The cryo-EM structure of Geo Cas9 in complex with gRNA (PDB: 8JTR) shows poor resolution of HNH. The Geo Rec2 domain from PDB: 8JTR (gray) is overlaid with our X-ray structure of Geo Rec2 (red, PDB: 9B72). ( B ) 1 H 15 N TROSY HSQC NMR spectrum of Geo Rec collected at 850 MHz. Overlays of this spectrum with resonances from spectra of Geo Rec1 (black) and Geo Rec2 (blue) demonstrate a structural similarity between the isolated subdomains and intact Geo Rec.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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: ( A ) Sequence alignment of Geo Rec2 and Spy Rec3. Conservedresidues are highlighted pink and are listed as consensus. ( B ) Overlay of the Geo Rec2 X-raycrystal structure (gray) with Spy Rec3 from full-length Spy Cas9 (pink, PDB: 4UN3). ( C ) Overlayof the Geo Rec1 structure derived from full-length Geo Cas9 modeled with Alphafold2 (gray)with the homologous portion of Spy Rec1and Spy Rec2 from Spy Cas9 (PDB: 4UN3) in red andorange, respectively.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

Techniques: Sequencing, Derivative 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: ( A, B ) Sites of selected mutations within Geo Rec2, K267, and R332, are highlighted as purple sticks directly facing the RNA and DNA modeled from Nme Cas9 (PDB ID: 6JDV), allowing for prediction of the binding orientation within Geo Cas9. NMR chemical shift perturbations caused by the K267E ( C ) or R332A ( D ) mutations are plotted for each residue of Geo Rec. Gray bars denote sites of line broadening, the blue bar denotes an unassigned region of Geo Rec 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 ) Geo Rec (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 Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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: ( A ) CPMG relaxation dispersion profiles of all residues with evidence of μs-ms motion, fit to a global k ex of 147±41 s –1 (WT Geo Rec2, left), 376±89 s –1 (K267E Geo Rec2, center), and 142±28 s –1 (R332A Geo Rec2, right) collected at 25 °C and 600 MHz. Residues are colored in accordance with . Relaxation dispersion profiles for individual resonances are shown in – . ( B ) Sites exhibiting CPMG relaxation dispersion in ( A ) are mapped to Geo Rec as blue spheres. Adjacent domains within the cryo-EM structure of Geo Cas9 are also shown. ( C ) Per-residue NMR order parameters of WT (black), K267E, and R332A (red, separate plots) Geo Rec.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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: ( A ) and truncated ( B ) Geo Cas9 gRNAs used in this work, based on the cryo-EM structure PDB: 8UZA. The 2D structure cartoons below each representation were predicted from sequence by the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) maintained by the Institute for Theoretical Chemistry, University of Vienna.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

Techniques: Cryo-EM Sample Prep, Sequencing

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: ( A ) CD spectroscopic unfolding profiles of WT Geo Cas9 (black) reveal a marked gRNA-dependent stabilization (red). The T m of each state is inset, as is the Δ T m . The same CD profiles for K267E Geo Cas9 ( B ) highlights a weaker stabilization and Δ T m upon gRNA binding, while those of R332A Geo Cas9 ( C ) show virtually no difference in T m between apo and RNP samples. ( D ) Fitted denaturation profile overlays of apo WT (black line), K267E (dashed line), and R332A (dotted line) Geo Cas9 (left) and RNP complexes (141nt gRNA, right). The red dashed line denotes the T m of the apo proteins, which are nearly identical. All data were fit as described in the Materials and Methods.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

Techniques: 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: ( A ) The structure of Geo Cas9 (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 i Geo Cas9 (lime green), all highlighted in surface representation. ( B ) Differential root-mean-square fluctuations (∆RMSF) of protein residues computed between WT Geo Cas9 and the K267E (red), R332A (blue), and double mutant (pink). ( C ) Distribution of protein-RNA contacts for WT and Geo Cas9 variants computed over the 6 μs simulation ensemble. ( D ) Comparison of gRNA binding free energy to the Rec domain in WT Geo Cas9 and variants. ( E ) Representative snapshots from MD simulations illustrating structural changes in Rec-gRNA association in WT Geo Cas9 (left) and variants (right).

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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: ( A ) Root-mean-square deviation (RMSD) distribution of WT (gray), K267E (red), R332A (blue), K267E/R332A double mutant (pink) and i Geo Cas9 (green). ( B ) Conformational changes in the HNH and Rec domains of the mutant systems (black arrows), compared to WT Geo Cas9. ( C ) Distribution of protein-DNA contacts for WT and mutants computed over the 6 μs ensemble. ( D ) Differential RMSD (ΔRMSF) of the protein residues computed between the WT Geo Cas9 and K267E (red) and i Geo Cas9 (green). ( E ) Distribution of protein-RNA contacts for WT, K267E and i Geo Cas9 computed over the 6 μs ensemble. ( F ) Comparison of binding free energy of RNA with Rec domain of Geo Cas9 between WT, K267E and i Geo Cas9.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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: In vitro cleavage assays indicate no significant difference in temperature-dependent activity between WT, K267E, and R332A Geo Cas9. ( A ) RNP concentration in each lane (left-to-right) is 100, 200, 300, 600, or 900 nM and 0 nM (control) at each temperature tested. Molecular weight markers on agarose gels (top-to-bottom) are 600 and 400 basepairs. ( B ) Graph quantifying the DNA band intensity measurements on the agarose gel ( A ) using ImageJ. Data plotted as mean ± SD of n=3 technical replciates. Figure 7—figure supplement 1—source data 1. Raw gel image of DNA cleavage by WT Geo Cas9, K267E Geo Cas9, and R332A Geo Cas9 at 37, 60, 75, and 85 °C. Figure 7—figure supplement 1—source data 2. Raw gel image of DNA cleavage by WT Geo Cas9, K267E Geo Cas9, and R332A Geo Cas9 at 37, 60, 75, and 85 °C, labelled.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

Techniques: In Vitro, Activity Assay, Concentration Assay, Control, Molecular Weight, Agarose Gel Electrophoresis

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: ( A ) Off-target in vitro cleavage assay with WT, K267E, R332A, and K267E/R332A Geo Cas9. ( B ) Off-target in vitro cleavage assay with WT and HiFi Spy Cas9. Data plotted as mean ± SD of n=3 technical replicates. Legend: WT = on-target DNA substrate at the mouse Tnnt2 gene locus. mm5-6 = off-target DNA with the 5th and 6th nucleotide mismatches from the PAM seed site. mm19-20 = off-target DNA with the 19th and 20th nucleotide mismatches from the PAM seed site. Full DNA sequences of the substrates for the Geo Cas9 and Spy Cas9 can be found in and , respectively.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

Techniques: In Vitro, Cleavage 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: ( A ) Representative MST-derived profiles of WT, K267E, and R332A Geo Cas9 binding to a Cy5-labeled full-length Tnnt2 gRNA. ( B ) Bar graph comparing K d values across n≥3 technical replicate samples are shown for Tnnt2 and 8UZA gRNA from a recent cryo-EM structure of Geo Cas9. *p<0.01.

Article Snippet: The full-length Geo Cas9 plasmid was acquired from Addgene (#87700), expressed in TB media and was expressed and purified as previously described ( ).

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