E box cx5 gel documentation system

3’-Fluorescein-labeled crRNAs 10 µM solutions were used for investigation of their resistance to nuclease digestion. crRNAs were incubated in 10% fetal bovine serum (Sigma, USA) in IMDM culture medium (Sigma, Burlington, MA, USA) for 1 h at 37 °C. After fixed times, 1 μL (10 pmol) aliquots of this mixture were taken and added to the stop buffer (8 M urea, 50 мM Na₂EDTA, 89 мM Tris-borate (pH 8.3), 0.1% SDS) at 0 °C. The reaction products were analyzed by gel electrophoresis in 15% polyacrylamide gel and visualized at 312 nm using the E-Box-CX5 gel documentation system (Vilber Lourmat, Collégien, France). The images were quantified using the Quantity One program (Bio-Rad, Hercules, CA, USA). The cleavage efficacy was calculated by the following equation: $$P_t=\left(P_0-P_{\infty }\right)·e^{\left(-K·t\right)}+P_{\infty }$$
where $P_t$ —fraction of non-cleaved crRNA at a certain moment of time, t—time in minutes, K—crRNA cleavage rate constant, $P_0$ —fraction of non-cleaved crRNA at the initial moment, $P_{\infty }$ —fraction of non-cleaved crRNA in unlimited time. The kinetic parameters of cleavage were determined from the cleavage curves using the GraphPad Prism 7.00 software.

The CNP-immobilized duplexes of PC-DNA with crRNA_Flu were irradiated with light at 365 nm for 0.5, 1, 2, 5, 10, 15, 30, and 60 min. The cleavage products were analyzed in denaturing 15% PAGE, followed by visualization using the E-Box-CX5 gel documentation system (Vilber Lourmat, France). The images were quantified using the Quantity One program (Bio-Rad, United States). The portion of the released crRNA_Flu was calculated using the Microsoft Excel software. Non-immobilized crRNA_Flu taken at the same quantity served as a control corresponding to the 100% of free RNA. The parameters were calculated in the GraphPad Prism 5.0.4.533 software package (Graph-Pad, United States) using the Equation (3): $$f_a=P_{st} ·\left(1-e^{k_{1}t}\right)$$
where $$f_a$$ is a portion of the released crRNA_Flu; $$P_{st}$$ is a portion of the released crRNA_Flu during the transition of the reaction to the stationary phase (the maximum degree of release); $$k_1$$ is the pseudo-first-order reaction constant; t is the reaction time.

The cleavage of the plasmid by the Cas9 protein in the presence of guide RNAs was analyzed by gel electrophoresis in 1% agarose gel in a TAE buffer (4 mM Tris, 3 mM CH₃COOH, 0.07 mM Na₂EDTA). The reaction mixture (10 μL) in the Quenching Buffer (250 mM Na₂EDTA, 1.2% SDS, 0.01% bromophenol blue and 30% glycerol) was applied to the gel. The DNA marker 1 kb (250 to 10,000 bp dsDNA ladder; SibEnzyme, Nowosibirsk, Russia) was used to compare the mobility of the cleavage products. The gel was stained with an ethidium bromide solution. The initial plasmid and the products of cleavage were visualized using the E-Box-CX5 gel documentation system (Vilber Lourmat, Marne-la-Valee, France). The images were quantified in the Quantity One program (Bio-Rad, Hercules, CA, USA). The portion of the plasmid cleavage was calculated by the following equation: $$N_{\Sigma }=\frac{I_{lin}+I_{rel}}{I_{lin}+I_{rel}+I_{superc}/k} ·100\%,$$
where $N_{\Sigma }$ is the total plasmid cleavage; $I_{lin}$ is the intensity of the band corresponding to the linear form of the plasmid; $I_{rel}$ is the intensity of the band corresponding to the relaxed form of the plasmid; $I_{superc}$ is the intensity of the band corresponding to the supercoiled form of the plasmid; k = 1.14 is the coefficient of staining efficiency of the supercoiled form of DNA relative to the relaxed form [33 (link)]. The efficacy of cleavage was determined using the Microsoft Excel software.