Despite being originally developed for gene editing, CRISPR-Cas systems have proven utility in disease diagnostics due to their ability to precisely target DNA. To overcome weak signaling capacities, CRISPR-Cas based sensors are often coupled with nucleic acid amplification; however, an alternative strategy is to engineer the reporter to have signal-generating properties. Current CRISPR-Cas12a assays equip reporters that rely on fluorescein (FAM)-Black Hole Quencher (BHQ) quenching and the trans cleavage of probe ssDNA by activated Cas12a to generate signal via unquenched FAM.
Jake Lesinski and Nathan Khosla from the deMello group at ETH Zurich have recently developed a new CRISPR-Cas12a assay reporter that provides for improved stability and shorter times-to-result. Notably, the reporter can be directly inserted into existing assays and works with clinical samples. The reported leverages Förster resonant energy-transfer (FRET) between donor and acceptor molecules to generate signal. Such an approach offers a number of advantages over BHQ reporters, including superior stability against degradation and faster cleavage rates by Cas12a. Additionally, ratiometric analysis enhances signal-to-noise ratios, reduces measurement uncertainty and leads to faster times-to-result. The team’s work highlights the impact a small change to the reporter can have on assay performance and poses the question of whether other CRISPR-Cas reporters could similarly benefit from dual-channel ratiometric signaling.
Written by Yukina Partington
Read the published article here.
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