A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity

Abstract

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120–145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi

“…The released chromophore spontaneously dimerizes and oxidizes into 5,5'-dibromo-4,4'-dichloro-indigo, producing a colorimetric clear-to-blue output that is visible to the naked eye. We achieve a lower limit of detection (picomolar) than the fluorophore quencher strategy first employed by Chen et al 15 , explore and give insight into the difference made by the oligo linker length 26,27 and show that our assay can be lyophilized and still maintain functionality. Overall, this tool would improve the diagnosis of enteric fever in the Global South by providing an affordable solution which does not require detection equipment.…”

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts

“…Trans-cleavage destroys the ssDNA probe and releases the label from the surface. In this case, the label can be a fluorescent molecule, an enzyme, a nanozyme, or another detectable molecule [14][15][16][17][18].…”