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CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli

Agnieszka Bogut

Agnieszka Bogut

Chair and Department of Medical Microbiology, Medical University of LublinLublin, Poland
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Bogut, Agnieszka
, 
Anna Kołodziejek

Anna Kołodziejek

Department of Animal, Veterinary, and Food Science, University of IdahoMoscow, USA
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Kołodziejek, Anna
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Scott A. Minnich

Scott A. Minnich

Department of Animal, Veterinary, and Food Science, University of IdahoMoscow, USA
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Minnich, Scott A.
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Carolyn J. Hovde

Carolyn J. Hovde

Department of Animal, Veterinary, and Food Science, University of IdahoMoscow, USA
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Hovde, Carolyn J.
  
07 gen 2025
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Categoria dell'articolo: Review
Pubblicato online: 07 gen 2025
Ricevuto: 17 giu 2024
Accettato: 04 nov 2024
DOI: https://doi.org/10.2478/aite-2025-0003
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© 2025 Agnieszka Bogut et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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.
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.
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.
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.

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

Turnaround time: 70 min

Sensitivity of detection on food products: 4.80 × 10°CFU/g

 Fluorescence Lee and Oh (2022)
LAMP-CRISPR/Cas12a ecs_2840R Detection of E. coli O157:H7 in spiked milk

Turnaround time: 60 min

Sensitivity of detection in milk:

7.4 × 10°CFU/mL following 3 h of cultivation;

7.4 × 102 CFU/mL without incubation

 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

Turnaround time: 45 min

Sensitivity of detection in artificially contaminated samples: >2.5 × 102 CFU/mL

 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

Turnaround time: 55 min

Sensitivity:

~1 CFU/mL and

1 × 102 CFU/mL for the fluorescence and the lateral flow assay, respectively

 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

Turnaround time: 50 min

Sensitivity: 17.4 CFU/mL

 Evanescent wave fluorescence biosensor Song et al. (2023)
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