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

Abstract

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 the nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity‐ and fluorescent protein dual‐tagged Csx30 protein substrate, we 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 with concentrations down to 25 pM, which could be further improved by approximately 15,000‐fold (~ 2 fM) through incorporating the 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 of various species, potentially providing an easily adaptable platform for clinical diagnosis.

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30

“…We could successfully detect and discern either one RNA variant or both variants in a single reaction, demonstrating multiplexed RNA detection (Figure C). To circumvent the need for a protein gel readout, a recent study fused a fluorescent protein to the C-terminal side of Csx30 . This allows measurement of the time-dependent increase in fluorescence intensity upon Csx30 cleavage, allowing picomolar detection sensitivity.…”

“…This provides distinct benefits for biotechnological applications that require multiplexed cleavage specificities, for which we provide a proof-of-principle in this study. By programing the Craspases to recognize specific target RNA of pathogens, such as COVID-19 variants, one could exploit this assay for point-of-care diagnostics. This is similar to the Cas13- and Cas12-based multiplexing assays, where multiple variants can be detected in one-pot reactions using effector orthologs and different RNA or DNA reporters. , An advantage of the Craspase multiplexing assay is its use of protein-based reporters, which may be useful when reporter stability is desired.…”

“…To circumvent the need for a protein gel readout, a recent study fused a fluorescent protein to the C-terminal side of Csx30. 12 This allows measurement of the time-dependent increase in fluorescence intensity upon Csx30 cleavage, allowing picomolar detection sensitivity.…”