Publication
Accelerated RNA detection using tandem CRISPR nucleases
Publisher:
Cold Spring Harbor Laboratory
Date:
24-03-2021
DOI:
10.1101/2021.03.19.21253328
Abstract: Direct, lification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental s les. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule 1,2 , but long reaction times h er sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal s les with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.