CRISPR–Cas based platforms for RNA detection: fundamentals and applications

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Abstract

The detection of RNA biomarkers is crucial for diagnosing many urgent diseases such as infections and cancer. Conventional RNA detection techniques such as RT-PCR, LAMP, and microarrays are effective, but often face limitations in terms of speed, sensitivity, and equipment demands. In recent years, CRISPR/Cas systems have emerged as versatile platforms for RNA detection, which offer high specificity, programmability, and adaptability across a wide range of diagnostic applications. This review first categorizes different CRISPR-based RNA detection systems according to the CRISPR effectors employed, including Cas13, Cas12, Cas14, Cas9, and newly characterized enzymes such as Cas7-11 and Cas10, detailing their mechanisms of target recognition, cleavage activity, and signal generation. The CRISPR detection platforms are coupled with or without pre-amplification steps to meet the different sensitivity needs. Preamplification-based systems integrate CRISPR with methods like RT-PCR and isothermal amplification to enhance sensitivity. In parallel, preamplification-free strategies, such as split-crRNA or split-activator systems, are gaining attention for their balanced assay performance and simplicity, which are especially attractive for point-of-care (POC) settings. Then, the diagnostic applications of these technologies are explored across two major domains: infectious disease detection and cancer biomarker identification via miRNAs, demonstrating the clinical potential of CRISPR-based RNA detection platforms. In addition, we explore ongoing challenges such as improving sensitivity in amplification-free formats, and developing field-deployable, cost-effective systems. The review concludes by outlining emerging trends and future directions in CRISPR-based RNA diagnostics, emphasizing their transformative potential in clinical settings.

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