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: Cell

Article Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex

doi: 10.1016/j.cell.2022.11.009

Figure Lengend Snippet:

Article Snippet: The ShTnsC gene was inserted into the 1S (Addgene #29659) plasmid to produce a construct carrying an N-terminal hexahistidine and hexahistidine-SUMO tag followed by a TEV cleavage site and the ShTniQ gene was cloned into the 1C (Addgene #29654) vector generating a construct carrying a hexahistidine-maltose binding protein (6xHis-MBP) tag followed by a TEV cleavage site.

Techniques: Recombinant, Mass Spectrometry, Electron Microscopy, Plasmid Preparation, Virus, Cloning, Software

Journal: Analytical Methods

Article Title: Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform

doi: 10.1039/c8ay02726a

Figure Lengend Snippet: Fig. 2 Development of Cas9 protein and activity detection assays. (a) Detection of Cas9 by capture antibody-functionalized silica microparticles and detection antibodies for S. pyogenes (2 pg mL1), S. aureus (44 pg mL1), S. thermophilus (31 pg mL1), and N. meningitidis (23 pg mL1) using chemiluminescence detection (b) Detection of S. pyogenes Cas9 by capture and detection antibodies have unchanged luminescence profiles regardless of biological matrix. (c) Detection of Cas9 by AcrIIC1-functionalized silica microparticles as a capture reagent. N. meningitidis Cas9 (32 pg mL1) and C. jejuni Cas9 (1293 pg mL1) were detected by species-specific antibodies, while G. stearothermophilus Cas9 (188 pg mL1) could be detected by cross-reactive S. aureus Cas9 antibodies. (d) Microparticle-immobilized fluorescence-based detection of S. pyogenes Cas9 nuclease activity in the presence of the substrate-specific AAVS1 guide RNA. Error bars represent standard deviation, points are average of four replicates, and the limits of detection are indicated in parentheses for each measurement.

Article Snippet: S. pyogenes wild-type, D10A, H840A, and dead Cas9 were overexpressed as His-MBP-TEV fusion proteins and puried as described previously.21 S. aureus and C. jejuni Cas9 were overexpressed and puried as described previously.21 Plasmids encoding G. stearothermophilus Cas9 (a kind gi of Jennifer Doudna,18 Addgene #87703) andN. meningitidis Cas9 (a kind gi of Erik Sontheimer,22 Addgene #71474) were expressed as described in their respective papers and puried just as S. aureus and C. jejuni Cas9 from above.

Techniques: Activity Assay, Standard Deviation

Journal: Analytical Methods

Article Title: Ultrasensitive multi-species detection of CRISPR-Cas9 by a portable centrifugal microfluidic platform

doi: 10.1039/c8ay02726a

Figure Lengend Snippet: Fig. 3 Simultaneous Cas9 protein and nuclease activity detection. (a) Detection of chemiluminescent Cas9 protein and fluorescent nuclease activity for wild type (wt), the D10A and H840A nickase mutants with one active site disrupted, and the nuclease D10A/H840A dead Cas9 (dCas9) at 0 nM and 10 nM of Cas9. (b) Detection of Cas9 protein and activity in human HEK293T cells transfected with on-target AAVS1 guide (AAVS1), off-target guide (scrambled), or an untransfected control. Error bars represent standard deviation, points are average of four replicates.

Article Snippet: S. pyogenes wild-type, D10A, H840A, and dead Cas9 were overexpressed as His-MBP-TEV fusion proteins and puried as described previously.21 S. aureus and C. jejuni Cas9 were overexpressed and puried as described previously.21 Plasmids encoding G. stearothermophilus Cas9 (a kind gi of Jennifer Doudna,18 Addgene #87703) andN. meningitidis Cas9 (a kind gi of Erik Sontheimer,22 Addgene #71474) were expressed as described in their respective papers and puried just as S. aureus and C. jejuni Cas9 from above.

Techniques: Activity Assay, Transfection, Control, Standard Deviation