Novel Application of Graphene Oxide in Conjunction with DNA Binding dCas9 for Detecting Antibiotic Resistance Genes

The frequent emergence of antibiotic resistance genes poses a stumbling block to prompt treatment of pathogen-originating diseases and therefore spurs the demand for a simple, rapid diagnostic device that can outcompete the time and cost of traditional methods such as PCR and cell culturing. The proposed method for detecting pathogenic DNA utilizes the binding properties of deactivated CRISPR-associated protein9 (dCas9) complexed with fluorescein-labeled single-guide RNA (sgRNA) and the fluorescence quenching properties previously demonstrated in graphene oxide (GO). For our assay, we designed sgRNAs to target specific 20 nucleotide sequences extracted from the tetracycline resistance (tetM) gene sequence and covalently modified the resulting constructs with a fluorescein derivative. We predicted that the dCas9 and sgRNA ribonucleoprotein complexes would interact with surface groups on the GO’s surface, bringing the fluorophores into GO’s quenching proximity. Upon binding of DNA, the dCas9 would undergo a conformational change, thereby disrupting the interactions and allowing the ribonucleoprotein and DNA complex to dissociate out of quenching proximity to result in the recovery of the fluorescence signal that can be measured using a plate reader. This method has been validated by predecessors in this lab, but as reported here, has undergone several optimizations to increase consistency and confidence in the results. Future studies will continue to optimize the assay for highly sensitive and specific detection of genomic and cell lysates such that the assay can be implemented as a simple and rapid diagnostic tool