Rapid screening of antimicrobial probiotics using CRISPR cascade

Animal bacterial infection is increasingly threatening human health. Here we report a nucleic acid amplification-free CRISPR genetic assay that allows to rapidly screen potential food-origin antimicrobial probiotics. The assay (termed CRISPRzyme assay) is based on a CRISPR-DNAzyme cascade, where the target gene sequentially activated Cas12a protein and DNAzyme, yielding a limit of detection of 62 CFU Vibrio parahaemolyticus, 86 CFU Salmonella Typhimurium, and 82 CFU Listeria monocytogenes. The elimination of nucleic acid amplification shortens processing time and operational complexity. The assay was used to rapidly screen antimicrobial probiotics by end-measurement of fluorescence of pathogenic bacteria. Particularly, it can estimate the in vivo antimicrobial effect due to its capacity for pathogen quantification in complex samples. We found that isolates of Bacillus and lactic acid bacteria separated from fermented food exhibited strong antimicrobial activity for fish pathogen, Vibrio parahaemolyticus, and identified surfactin as the key antimicrobial component. The CRISPRzyme assay could ease antimicrobial probiotics screening, and constitutes a new tool for combatting pathogenic bacterial contamination and infection.