Suppression of Antimicrobial Resistance in MRSA Using CRISPR-dCas9

Karissa Wang; Matthew Nicholaou

doi:10.29074/ascls.30.4.207

Article Figures & Data

Figures

Tables

Download figure
Open in new tab
Download powerpoint
Figure 1.
RT-qPCR amplification plots, melt curves, and standard curves. Two independent RT-qPCR experiments were run, with the standard curve in triplicate each time. mecA data is shown in red and 16s data in blue. a) The amplification plot shows that desired precision and amplicon specificity were achieved. b) A smooth, single-peaked melt curve shows satisfactory amplicon specificity for both 16s RNA and mecA. c) The standard curves show log concentrations of MRSA mRNA plotted against quantification cycle values. The slopes are used to describe the efficiency of each target, where an ideal efficiency would yield a slope of −3.3.
Download figure
Open in new tab
Download powerpoint
Figure 2.
Susceptibility testing on CRISPR-dCas9 treated isolates. a) Cefoxitin disk diffusion testing was performed in quadruplicate. The dark line represents the breakpoint between resistant and susceptible isolates. b) Oxacillin Microbroth serial dilutions performed in duplicate confirmed results from the disk diffusion test. Plasmid S43 reduced cefoxitin resistance, but did not achieve in vitro susceptibility and plasmid S46 had no effect (data not shown).
Download figure
Open in new tab
Download powerpoint
Figure 3.
CRISPR-dCas9 system significantly decreased mecA expression in MRSA. mecA mRNA expression was significantly reduced in S43 CRISPR-dCas9 transformed MRSA (p<0.0005). mecA expression was reduced by 77% to 0.23 of its original value, as compared to a mock-transformed control. Fold differences were calculated using the relative standard curve method. Shown above are fold differences ± standard error of the mean.

Tables

Figures

View popup

Table 1.

RT-qPCR data and fold difference calculations. Two independent RT-qPCR runs were performed, each with two RNA extractions that each tested for 16s and mecA expression levels in triplicate. Using the comparative quantification standard curve method, the fold difference in bacterial gene expression was given by the ratio of the efficiency of the target gene raised to the ΔC_t of the target to the efficiency of the normalizer gene raised to the ΔC_t of the normalizer. Efficiencies were calculated from the slopes of the standard curves. Calculations showed an average fold difference of 0.230, meaning that the S43 CRISPR-dCas9 treated MRSA had a significant decrease of 77% in mecA gene expression.