Gel imager system

SDS-PAGE was used to analyze the effects of CAN on MRSA bacterial protein levels. MRSA pretreatment was the same as above. CAN was added to the bacterial suspension to reach the final concentrations of 25 and 50 μM. The configured samples were transferred to a 6-well plate at 37 °C with 2 mL per well and cultured for 12 h. Then, the samples were collected and centrifuged at 10,000× g at 4 °C for 5 min. The supernatants were discarded, and the bacteria pellets were washed twice with PBS. The bacterial lysates were added according to the respective OD values, i.e., (OD − OD_0h) × 200 μL/(OD_ctrl − OD_0h). After adding the bacterial lysate, ultrasonic cracking was performed for 10 min per sample, followed by centrifugation at 8000× g at 4 °C for 10 min. Next, the supernatants were collected, and a loading buffer of 20 μL was added to the 80 μL supernatant. The loading buffer contained 100 mmol/L Tris-HCl pH 6.8, 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerine, and 200 mmol/L dithiothreitol (DTT). The prepared samples were then boiled at 100 °C for 10 min, cooled on ice, and analyzed using SDS-PAGE. After electrophoresis, the protein bands were stained with Coomassie Brilliant Blue R-250 and then decolorized to obtain the separated protein bands. The results of SDS-PAGE were photographed with a Gel Imager System (Thermo Fisher, Shanghai, China).

The Cas13a cleavage reaction consisted of 25 mM Tris HCl (PH 7.4), 9 mM MgCl₂, 200 ng Cas13a (Genscript, Cat# Z03486), 200 ng crRNA, 500 ng target RNA, and DEPC water added to the total mixture volume of 20 μL. The mixture was incubated at 37 °C for 30 min and heated at 70 °C for 10 min to denature RNA. The cleavage mixture was loaded onto 12% TBE-Urea Gels with 120 V for 60 min and analyzed on a gel imager system (Thermo Fisher Scientific). As the fluorescent cleavage assay, the Cas13a/crRNA reaction included 25 mM Tris HCl, 9 mM MgCl₂, 100 ng Cas13a, 20 ng crRNA, 10 mM rNTP (NEB, Shanghai, China, Cat# N0466S), 15 U T7 RNA polymerase (NEB, Cat# M0251S), 8 U RNA inhibitor (NEB, Cat# M0314S), 0.25 μM FAM-labeled ssRNA reporters, 20 ng DNA template, 50 ng total human RNA (Thermo Fisher Scientific, Cat# 4307281), and DEPC water added to the total mixture volume of 20 μL. The fluorescent signal of the Cas13a reaction was detected by the 7500 fast Real-Time PCR Systems (Thermo Fisher Scientific, USA) at 37 °C for 40 min and analyzed with the 7500 Fast Software v2.3. By observing the TBE-Urea gels and fluorescence values, we determined the activity and specificity of the mutant-crRNA.

After 8 weeks of intragastric administration, the total protein of the femur tissues was extracted and quantified. The protein sample was mixed with the loading buffer and denatured in a boiling water bath. The denatured protein samples were separated by gel electrophoresis and blocked with 5% skim milk powder after membrane transference. Primary antibody was added to the membrane for incubation at 4℃ overnight. HRP Goat Anti-Rabbit IgG (AS014, Abclonal, China) was added after washing with PBS and incubated at room temperature for 1 h. ECL luminescent solution (24,580, Thermo Fisher, USA) was used for luminescence, and then the protein bands were exposed to Gel Imager System. Primary antibodies Runx2 (A2851), Osterix (A18699), and GAPDH (A19056) were all purchased from Abclonal, China.