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

Journal: Analytical methods : advancing methods and applications

Article Title: Ultrasensitive Multi-Species Detection of CRISPR-Cas9 by a Portable Centrifugal Microfluidic Platform

doi: 10.1039/C8AY02726A

Figure Lengend Snippet: (a) Detection of Cas9 by capture antibody-functionalized silica microparticles and detection antibodies for S. pyogenes (2 pg/mL), S. aureus (44 pg/mL), S. thermophilus (31 pg/mL), and N. meningitidis (23 pg/mL) 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/mL) and C. jejuni Cas9 (1293 pg/mL) were detected by species-specific antibodies, while G. stearothermophilus Cas9 (188 pg/mL) 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: 21 Plasmids encoding G. stearothermophilus Cas9 (a kind gift of Jennifer Doudna 18 , Addgene #87703) and N. meningitidis Cas9 (a kind gift of Erik Sontheimer 22 , Addgene #71474) were expressed as described in their respective papers and purified just as S. aureus and C. jejuni Cas9 from above.

Techniques: Fluorescence, Activity Assay, Standard Deviation

Journal: Analytical methods : advancing methods and applications

Article Title: Ultrasensitive Multi-Species Detection of CRISPR-Cas9 by a Portable Centrifugal Microfluidic Platform

doi: 10.1039/C8AY02726A

Figure Lengend Snippet: (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). (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: 21 Plasmids encoding G. stearothermophilus Cas9 (a kind gift of Jennifer Doudna 18 , Addgene #87703) and N. meningitidis Cas9 (a kind gift of Erik Sontheimer 22 , Addgene #71474) were expressed as described in their respective papers and purified just as S. aureus and C. jejuni Cas9 from above.

Techniques: Activity Assay, Transfection, Control, Standard Deviation