Break

CRISPR-Cas nucleases have fundamentally been understood as RNA-guided systems, critical for applications in genome editing and diagnostics. The most commonly known CRISPR-Cas system is Cas9 which is commonly used in gene editing due to its high cleavage efficiency. Another commonly used CRISPR-Cas system is Cas12, which are novel enzymes used for diagnostic purposes due to its Trans-cleavage activity. Trans-cleavage is the process when a Cas-crRNA complex binds to a DNA target sequence, resulting in collateral cleavage of ssDNA. If ssDNA with a fluorophore and a quencher are exposed to such cleavage, fluorescence can be measured to determine the presence of a target sequence. This study explores the potential of Cas12 family members to use synthetic small DNA sequences to guide RNA detection for RNA detection. Our findings reveal some Cas12 proteins can activate trans-cleavage in the presence of RNA targets using short DNA “guides”, providing a novel mechanism for RNA targeting. We observed that Cas12 could use a spacer-only RNA guide without the traditional scaffold sequence for ssDNA detection. This led to the hypothesis that short synthetic DNA sequences could potentially be employed to detect RNA targets. Experimentation with synthetic DNA guides revealed that adding a loop at the 3’-end of a complementary DNA (cDNA) substantially increased trans-cleavage activity for RNA detection. We demonstrated the efficacy of these guides at detecting a variety of miRNA biomarkers, such as miR-21, miR-122, and miR-155, as well as longer viral RNA sequences from HIV. The broad applicability of DNA guides across different CRISPR-Cas systems highlights their potential in molecular diagnostics. Our results underscore the versatility and cost-effectiveness of DNA guides, offering a transformative approach to nucleic acid detection and diagnostic tool development.