Detecting Disease-Associated Nucleic Acid Biomarkers Using Split-CRISPR-Cas12a Assay and Plasmid Reporter Read Out

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play essential roles in gene regulation and have been recognized as valuable biomarkers for diverse diseases, including cancer and neurological disorders. However, their short length, low abundance, and sequence similarity make sensitive and specific detection challenging. CRISPR-based diagnostics have recently emerged as a promising alternative to conventional miRNA detection methods, providing high specificity, programmability, and the potential for amplification-free workflows.

While Cas13a systems have been explored for direct RNA sensing, they face notable limitations, including reliance on unstable RNA reporters prone to RNase degradation, high reagent cost, and limited commercial availability. To address these challenges, this work presents two CRISPR-Cas12a–based strategies for direct miRNA detection: one employing a split activator design and the other utilizing a split crRNA system. Both approaches enable RNA sensing without reverse transcription or pre-amplification, thus simplifying the workflow and improving robustness compared to traditional Cas12a assays.

Our comparative analysis demonstrated that the split crRNA configuration achieved superior sensitivity, reaching femtomolar-level limits of detection. Furthermore, we introduced a plasmid-based length reporter as a novel, label-free alternative to conventional fluorescent ssDNA reporters. This plasmid reporter substantially reduces assay time and cost while maintaining high analytical performance.

The integration of the split crRNA design with the plasmid-based reporter establishes a rapid, cost-efficient, and sensitive CRISPR-Dx platform for miRNA detection. This strategy enhances the accessibility of CRISPR diagnostics and offers a robust framework for future clinical and point-of-care applications.