Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems

RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we have established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs at concentrations down to 25 pM, which could be further improved approximately 15 000-fold (ca. 2 fM) by incorporating a recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems from various species, thus potentially providing an easily adaptable platform for clinical diagnosis.