MULTIPLE GARNS FOR ENHANCED GENE DETECTION USING CRISPR-CAS12A

Abstract

CRISPR diagnostic systems have shown great potential for detecting SARS-CoV-2, but improvements in sensitivity, specificity, and turnaround time are needed. Typically, a gRNA with CRISPR-Cas12a is used to detect the target DNA and activate its collateral activity, which is then used to cleave a reporter probe. Here, we demonstrate that the parallel use of two or three gRNAs accelerates the probe cleavage rate by up to 4.5 times compared to individual gRNAs. This synergistic effect is related to the parallel activation of CRISPR-Cas12a and the individual activity of the gRNAs. This observation led to a system that, using two gRNAs, detected the SARS-CoV-2 RNA gene N at up to 10 viral copies after prior amplification with RT-LAMP.The proof of concept of the system was demonstrated with 100% detection of positive and negative clinical samples in ~25 minutes using the fluorescence plate reader and ~45 minutes for lateral flow test strips.