Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline

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Abstract

Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 μM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.
Original languageEnglish (US)
JournalFrontiers in Bioengineering and Biotechnology
Volume11
DOIs
StatePublished - Jan 19 2023

Bibliographical note

KAUST Repository Item: Exported on 2023-01-23
Acknowledgements: This work is supported by KAUST baseline funding to MM. We would like to thank members of the Genome Engineering and Synthetic Biology Laboratory at KAUST for their critical discussion of this work.

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