An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Detection of RNA

Abstract

Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.

CRISPR-Powered Strategies for Amplification-Free Diagnostics of Infectious Diseases

“…These bead-bound targets are then placed into the femtoliter-sized microwells to enrich the target, save the time of sample pretreatment, limit the fluorescence spread, and enhance the local signal intensity. As a result, dCRISPR significantly improves the sensitivity of detecting SARS-CoV-2 to an LOD of 2 aM (Figure C) . In a similar microwell-based assay utilizing the same CRISPR/Cas system and microwell size, the reason behind the higher sensitivity achieved by dCRISPR compared to open-SATORI is yet to be further investigated.…”

“…Utilizing signal enrichment, this method achieved a remarkable sensitivity of 6.5 aM, representing a 1000-fold improvement over the previous SATORI assay (Figure B) . In a related study, considering a previous study that demonstrated that the droplet cannot react stably due to factors such as surface tension, Jiang’s group developed an innovative approach called dCRISPR . This method involves coating a biotinylated DNA probe onto the surface of magnetic beads, which are utilized to capture the 3′ end of the target molecule.…”

“…An Integrated Amplification-Free Digital CRISPR/Cas-Assisted Assay for Single Molecule Dtection of RNA. ACS Nano 2023 , 17 , 7250–7256 (ref ). Copyright 2023 American Chemical Society.…”

“…121 In a related study, considering a previous study that demonstrated that the droplet cannot react stably due to factors such as surface tension, Jiang's group developed an innovative approach called dCRISPR. 126 This method involves coating a biotinylated DNA probe onto the surface of magnetic beads, which are utilized to capture the 3′ end of the target molecule. These bead-bound targets are then placed into the femtoliter-sized microwells to enrich the target, save the time of sample pretreatment, limit the fluorescence spread, and enhance the local signal intensity.…”

Digital Recombinase Polymerase Amplification, Digital Loop‐Mediated Isothermal Amplification, and Digital CRISPR‐Cas Assisted Assay: Current Status, Challenges, and Perspectives

“…Recent studies on amplification‐free digital CRISPR/Cas detection have offered insights for further enhancing the efficiency of one‐pot detection. [ 61,62,129,130 ] Strategies such as reducing compartment volume to concentrate templates and amplification products can expedite pre‐amplification time required to generate sufficient templates. [ 61,131 ] Additionally, optimizing the reaction conditions can enhance Cas protein activity and designing multiple CrRNAs to activate more Cas proteins can effectively shorten the signal generation time.…”