ABSTRACT
There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. 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, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.
Mechanism of CRISPR-Cas12a, and the signal output produced by enzyme trans-cleavage and the release of LacZ. 1) Initial set-up of inactivated system. 2) DNA isolation and amplification. 3) DNA addition to the biosensor. 4) CRISPR recognition of the DNA target and indiscriminate cleavage of all DNA. 5) LacZ release to the supernatant. 6) Transfer to an X-Gal environment. 7) X-Gal cleavage by LacZ and chromophore oxidation to the intensive blue indigo [illustration created with BioRender.com].
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- Cas12a
- CRISPR-associated protein 12a
- CRISPR
- clustered regularly interspaced short palindromic repeats
- dsDNA
- doublestranded DNA
- FQ
- fluorophore-quencher ssDNA
- gRNA
- guide RNA
- HRP
- horseradish peroxidase
- HPLC
- high-performance liquid chromatography
- IVT
- in vitro transcription
- LacZ
- β-galactosidase
- LB
- luria broth
- MALDI-TOF
- matrix assisted laser desorption ionization-time of flight mass spectrometry
- NAT
- nucleic acid test
- POC
- point-of-care
- ROC
- receiver operating characteristic
- ssDNA
- single-stranded DNA
- X-Gal
- 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside
