Origin 2020b

Stock standard solution of IgG with a concentration of 1.0 × 10⁻⁵ g/mL was prepared in PBS at pH 7.4 and stored in a freezer at −20 °C until use. By diluting the stock solution, the concentration range of the standard IgG solution was changed from 1.0 × 10⁻¹⁰ to 1.0 × 10⁻⁶ g/mL and then stored at 4 °C for future use. The IgG was injected into the sensing chip from low concentration to high concentration during detection and the response was monitored and recorded instantly. The result of three replications of all data was the average ± standard deviation (SD), and MATLAB 2020b (MathWorks) and Origin 2020b (OriginLab) were used for statistical analysis. The signal response and concentration were drawn to obtain the calibration curve.

To prepare RI samples, 6.8% to 41.7% sucrose was dissolved in DI water to obtain the aqueous solutions of 1.343, 1.353, 1.363, 1.373, and 1.383 RI. The RI was measured and confirmed by a refractometer. The stock standard solution (100 g/mL) was diluted with PBS and stored in a freezer at −20 °C. The samples were prepared and used in the same week to avoid inactivated IgG or streptavidin inducing inspection errors. The prepared IgG or streptavidin standard solution had a 1 × 10⁻¹⁰ to 1 × 10⁻⁶ g/mL concentration range. It was stored at 4 °C for further usage. Before quantitative testing the analyte, the PBS buffer of pH 7.4 was injected into the sensing chip as a baseline. A low-concentration to high-concentration detection was performed to obtain the corresponding signal responses. The test was repeated three times for each data point, and the data were represented by an average value and standard deviation (mean ± SD). Finally, Origin 2020b (OriginLab, Northampton, MA, USA) was used for statistical analysis. A linear relationship between signal response and concentration was drawn to obtain the calibration curve.

The reporter 293T cells were seeded into 96-well plates at a density of 10,000 cells per well and cultured overnight before the addition of DMSO alone or various compounds (in DMSO solution) at appropriate concentrations (0.125–100 μM) with or without 2 μg/mL doxycycline for an additional 72 h of incubation. The cells were then harvested for analysis with the CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) or analyzed by western blot with antibodies as indicated. The results were plotted in the GraphPad Prism 6.0 software or Origin 2020b (OriginLab, Northampton, MA, USA). The inhibiting efficiency in reporter 293T cells was calculated using the following equation:

where E’ is the inhibiting efficiency in reporter 293T cells, and P₀, P₁, and P₂ are the GFP positive cell ratio of the uninduced group, Dox and DMSO treated control group, and Dox and compound treated group, respectively.

The MST method was carried out similar to the previous report [34 (link),39 (link)]. The binding ability of the compounds to SpCas9 in different states was tested by Monolith NT.115 (NanoTemper Technologies, Munich, Germany). The SpCas9-EGFP fusion protein or the SpCas9-EGFP/gRNA RNP complex (protein: gRNA = 1:1) was diluted with a binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 10 mM MgCl₂) to make a final concentration of 200 nM. The protein diluent was mixed with the compound diluent in equal volume. The mixture was kept at room temperature for 10 min. Then the sample was loaded into the NanoTemper standard capillaries. All experiments were carried out at 25 °C, 80% MST power, and 50% LED power. The data were analyzed with the NanoTemper MO affinity analysis software (NanoTemper Technologies, Munich, Germany). The raw data were then exported and plotted with Origin 2020b (OriginLab, Northampton, MA, USA).

Data represent the mean ± standard deviation. Statistical significance was calculated using one-way analysis of variance followed by Tukey’s post hoc test using the Origin 2020b software (Origin Lab, Northampton, MA, USA). The data were considered statistically significant at p < 0.05.

Statistical analysis was performed in Origin 2020b (OriginLab, Northampton, MA, USA). The Shapiro–Wilk- and F-test of OriginPro was used to ensure normal distribution and equal variance of the dataset, followed by a univariate ANOVA (including the post hoc Bonferroni test). p-Values of 0.05 (* and letters), 0.01 (^**) and 0.001 (^***) were considered to indicate significant, strongly significant and highly significant differences as indicated in the individual experiments. There were no non-normal distributed data.

Data represent mean ± standard deviation (SD). Statistical significance was calculated using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test, using the Origin 2020b software (Origin Lab, Northampton, MA, USA). Data were considered statistically significant at p < 0.05.