Towards Accessible Diagnostics: Developing CRISPR-Based Colorimetric Tools for Low- and Middle-Income Countries

There is a critical need to implement a sensitive and specific point-of-care biosensor that addresses the instrument constraints and manufacturing challenges faced in under-resourced contexts. While limited financial capital and educational resources are limiting factors, the barriers to implementing effective healthcare systems are rooted in a far more complex set of interdependent issues. These include illness, unreliable supply chains, lack of investment, significant corruption, and minimal research infrastructure, among many others. Ultimately, these factors lead to a self-perpetuating cycle that sustains low levels of research-translation and, in turn, lack of accessible and reliable diagnostics, further exacerbating disease burdens. This project aims to adapt CRISPR-Cas12 technology into a robust, easily manufacturable, and virtually equipment-independent diagnostic test suitable for low- and middle-income countries. Enteric fever detection is explored given its highly contagious nature and prevalence of infection in low-resource contexts. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden and increase antimicrobial resistance. Due to their reported programmability and temperature versatility, CRISPR-Cas12a reactions were selected as the test platform. Design, production and screening of 48 gRNAs specific for S. Typhi were conducted considering various reaction parameters. The results were compared to existing gRNA prediction models. The findings revealed significant variability in gRNA performance, highlighting the limitations of current sequence-based prediction tools and the importance of empirical validation in reaction-specific contexts. To enhance accessibility and reduce dependence on laboratory equipment, the standard fluorescent CRISPR-Cas12a reaction was modified to create a colorimetric one. This was achieved by immobilising an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. This method demonstrated a lower limit of detection than standard fluorescent assays while remaining active for four weeks after lyophilisation at ambient temperature, facilitating the possibility of shipment without cold chain, significantly reducing deployment costs. Further simplification of this colorimetric two-step reaction was explored by replacing the full β-galactosidase with its split form. While technical challenges remain, this approach represents a meaningful step toward a fully integrated, low-cost, and locally manufacturable CRISPR diagnostic.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Pascual Garrigos, Ana

Advisor dc:contributor.advisor

• Molloy, jenny

• CRISPR-Cas12
• Colorimetric
• B-Galactosidase
• DNA
• Biosensor

Licence

• https://www.repository.cam.ac.uk/bitstreams/51668713-7523-4125-9cb4-490db4f941ad/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.122059

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/390506