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

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

Archivum Immunologiae et Therapiae Experimentalis
Volume 73 (2025): Issue 1 (January 2025)
By: Agnieszka Bogut,  Anna Kołodziejek,  Scott A. Minnich and  Carolyn J. Hovde  
Open Access
|Jan 2025

Figures & Tables

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

MethodologyTarget geneApplicationAssay characteristicsMethod of detection of the reaction productReferences
LAMP-CRISPR/Cas12a (using filtration enrichment)stx2Detection of E. coli O157:H7 in spiked romaine lettuce

  • Turnaround time: 70 min

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

FluorescenceLee and Oh (2022)
LAMP-CRISPR/Cas12aecs_2840RDetection 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

FluorescenceWang et al. (2024)
tHDA-CRISPR/Cas12a (combined with the filter concentration method)stx2Detection of E. coli O157:H7 in spiked fresh salad mixSensitivity of detection in food products: 103 CFU/gFluorescenceKim et al. (2023)
RAA-CRISPR/Cas12arfbEDetection of E. coli O157:H7 in spiked and ground beef samplesTurnaround time: 30 min (after 4 h enrichment in ground beef samples spiked with 9.0 CFU/25 g of E. coliFluorescenceFang et al. (2022)
CRISPR/Cas12a/RPArfbEDetection 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 chromatographyLuo et al. (2024)
MIRA/CRISPR/Cas12a (combined with the metal organic framework immunomagnetic beads enrichment)rfbEDetection of E. coli O157:H7 in ground beefSensitivity of target detection in ground beef: 14 CFU/mL (after 4 h of culture through Metal Organic Framework immunomagnetic beads enrichment)FluorescenceWang et al. (2021)
RAA/CRISPR/Cas12arfbEDetection 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 assayZhu et al. (2023)
HCR-CRISPR/Cas12arfbEDetection of E. coli O157:H7 in spiked environmental water samples

  • Turnaround time: 50 min

  • Sensitivity: 17.4 CFU/mL

Evanescent wave fluorescence biosensorSong et al. (2023)
DOI: https://doi.org/10.2478/aite-2025-0003 | Journal eISSN: 1661-4917
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Language: English
Submitted on: Jun 17, 2024
|
Accepted on: Nov 4, 2024
|
Published on: Jan 7, 2025
Published by: Hirszfeld Institute of Immunology and Experimental Therapy
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
EHEC,
CRISPR/Cas,
Cas9,
Cas12,
Probiotics,
O157:H7
Related subjects:
Medicine,
Basic medical science,
Biochemistry,
Immunology,
Clinical medicine,
Clinical medicine, other,
Clinical chemistry

© 2025 Agnieszka Bogut, Anna Kołodziejek, Scott A. Minnich, Carolyn J. Hovde, published by Hirszfeld Institute of Immunology and Experimental Therapy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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