Versatile and portable Cas12a-mediated detection of antibiotic resistance markers

Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results readout (Fluorescence vs. FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.