Fig 1.
The natural CRISPR/Cas9 antiviral defense system. The figure was prepared using the BioRender.com. Cas, CRISPR-associated proteins; CRISPR, clustered regularly interspaced short palindromic repeats; pre-crRNA, precursor crRNA; RNaseIII, endoribonuclease III; sgRNA, single-guide RNA; tracrRNA, trans-activating small RNA.
Fig 2.
The working principle of Cas9. Active ribonucleoprotein complex is formed by Cas9 protein and sgRNA, a hybrid of crRNA and tracrRNA. Cas9 contains two NUC domains: RuvC and HNH. The RuvC domain cleaves the protospacer sequence on the non-complementary strand. The HNH NUC domain cleaves a single strand containing 20-nt homology to the mature crRNA. G-rich (NGG) PAM follows immediately 3′- of the crRNA complementary sequence and is required for the cleavage. Site-specific DSBs in the target sequence with blunt ends are formed. The figure was prepared using the BioRender.com. Cas, CRISPR-associated proteins; crRNA, CRISPR RNA; DSBs, double strand breaks; NUC, nuclease; PAM, protospacer-adjacent motif; sgRNA, single-guide RNA; tracrRNA, trans-activating small RNA.
Fig 3.
The working principle of Cas12a. Cas12a has a single RuvC NUC domain. TTTV PAM and sequentially cleaves the non-targeting strand. The cleavage site in the targeting strand is defined by the spacer region in the crRNA. The DNA DSBs with sticky ends and a 4 or 5-nt 5′ overhang are formed. The figure was prepared using the BioRender.com. Cas, CRISPR-associated proteins; crRNA, CRISPR RNA; DSBs, double strand breaks; NUC, nuclease; PAM, protospacer-adjacent motif; TTTV, The enzyme recognizes T-rich.
Table 1.
CRISPR/Cas-based assays used for the identification of EHEC in food products of different origins
| Methodology | Target gene | Application | Assay characteristics | Method of detection of the reaction product | References |
|---|---|---|---|---|---|
| LAMP-CRISPR/Cas12a (using filtration enrichment) | stx2 | Detection of E. coli O157:H7 in spiked romaine lettuce |
| Fluorescence | Lee and Oh (2022) |
| LAMP-CRISPR/Cas12a | ecs_2840R | Detection of E. coli O157:H7 in spiked milk |
| Fluorescence | Wang et al. (2024) |
| tHDA-CRISPR/Cas12a (combined with the filter concentration method) | stx2 | Detection of E. coli O157:H7 in spiked fresh salad mix | Sensitivity of detection in food products: 103 CFU/g | Fluorescence | Kim et al. (2023) |
| RAA-CRISPR/Cas12a | rfbE | Detection of E. coli O157:H7 in spiked and ground beef samples | Turnaround time: 30 min (after 4 h enrichment in ground beef samples spiked with 9.0 CFU/25 g of E. coli | Fluorescence | Fang et al. (2022) |
| CRISPR/Cas12a/RPA | rfbE | Detection of E. coli O157:H7 in spiked romaine lettuce and in natural food products |
| Fluorescence and lateral flow chromatography | Luo et al. (2024) |
| MIRA/CRISPR/Cas12a (combined with the metal organic framework immunomagnetic beads enrichment) | rfbE | Detection of E. coli O157:H7 in ground beef | Sensitivity of target detection in ground beef: 14 CFU/mL (after 4 h of culture through Metal Organic Framework immunomagnetic beads enrichment) | Fluorescence | Wang et al. (2021) |
| RAA/CRISPR/Cas12a | rfbE | Detection of E. coli O157:H7 in spiked skim milk and drinking water |
| Fluorescence and the lateral flow assay | Zhu et al. (2023) |
| HCR-CRISPR/Cas12a | rfbE | Detection of E. coli O157:H7 in spiked environmental water samples |
| Evanescent wave fluorescence biosensor | Song et al. (2023) |
[i] CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated proteins; HCR, hybridization chain reaction; LAMP, loop-mediated isothermal amplification; MIRA, multienzyme isothermal rapid amplification; PMNT, cationic-conjugated polythiophene derivative; RAA, recombinase aided amplification; RPA, recombinant polymerase amplification; tHDA, thermophilic helicase-dependent amplification.