Carbon Quantum Dots Conjugated CRISPR-cas9-gRNA Antimicrobials Against Antibiotic Resistant Uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common bacterial infections. UTI, although treatable, is now becoming increasingly tough to control because of rampant antimicrobial resistance in the Enterobacteriaceae family, particularly inEscherichia coli. Due to increased resistance, there is an exigency to develop novel therapeutic targets to combat these resistance strains. Clustered regularly interspaced short palindromic repeats (CRISPR)-technology has emerged as an important tool for gene editing based therapeutics.

Here, we report an efficient gene editing strategy based on direct delivery of Cas9 and gRNA into Uropathogenic E. coli (UPEC) isolate. P-fimbriae plays major role in bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. We have covalently conjugated cas9 protein with CQD’s using EDC/NHS chemistry and targeted papG gene by synthesizing sequence specific complimentary gRNA. The targeting efficiency was confirmed by performing adherence assay, biofilm assay, flow cytometry and qRT-PCR.

There is a significant reduction of adherence ability of papG mutant strain as compared to control observed through microscopy. Also, significant reduction in biofilm formation (p-value < 0.001) was noticed in treated sample (0.565 ± 0.003) vs control (1.020± 0.008). The relative qRT-PCR data of knockdown strains (p<0.05) also revealed the dramatic decrease in the expression of papG gene, taking 16s rRNA gene as an endogenous control for normalization. The same result was observed from flow cytometry and SEM analysis.

Hence, we conclude that CQD-CRISPR-Cas9-gRNA could be a novel therapeutic strategy for the eradication or treatment of antibiotic resistant pathogenic E. coli-based pathologies.