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Related Experiment Video

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Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi.

Ana Pascual-Garrigos1, Beatriz Lozano-Torres1, Akashaditya Das2

  • 1Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB3 0AS, United Kingdom.

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|February 6, 2025
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Summary

This study introduces a new CRISPR-Cas12a biosensor for enteric fever, offering a naked-eye colorimetric readout. This affordable diagnostic tool bypasses the need for complex equipment, improving detection in resource-limited settings.

Keywords:
CRISPR-Cas12aDNAbiosensorcolorimetricβ-galactosidase

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Point-of-Care Testing

Background:

  • Enteric fever is a prevalent infection in low- and middle-income countries, often misdiagnosed due to ambiguous symptoms.
  • Lack of rapid, accurate, and affordable diagnostics exacerbates disease burden and contributes to antibiotic resistance.

Purpose of the Study:

  • To develop a sensitive and specific point-of-care (POC) biosensor for enteric fever.
  • To overcome instrument limitations and manufacturing challenges in resource-constrained environments.
  • To create a colorimetric readout for CRISPR-Cas12a assays.

Main Methods:

  • Developed a CRISPR-Cas12a readout module utilizing a β-galactosidase (LacZ) enzyme linked to an oligonucleotide.
  • Immobilized the enzyme-oligo complex, which is cleaved by target-activated CRISPR-Cas12a.
  • Used X-Gal substrate for a visible blue color change upon enzyme release, indicating DNA target presence.

Main Results:

  • Achieved a colorimetric output visible to the naked eye, acting as a signal amplifier for CRISPR assays.
  • Demonstrated detection of amplified bacterial genomic DNA with a lower limit of detection than standard fluorescent assays.
  • Confirmed enzyme activity stability for 4 weeks post-lyophilization, enabling transport without cold chain.

Conclusions:

  • The developed CRISPR-Cas12a readout module provides an equipment-free, affordable, and sensitive diagnostic method.
  • This technology has the potential to significantly reduce deployment costs and improve enteric fever diagnosis in resource-limited settings.
  • The lyophilized format enhances usability and stability for field deployment.