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

Chembiochem. 2023 Sep 14:e202300401. doi: 10.1002/cbic.202300401. Online ahead of print.ABSTRACTRNA-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 plat...
Source: Chembiochem - Category: Biochemistry Authors: Source Type: research