CRISPR-AD: Combinational Detection of Blood Protein and miRNA with Digital CRISPR-Based Assay Enable to Improve the Diagnostic Performance of Alzheimer’s Disease

Blood-based biomarkers present a noninvasive approach for detecting and assessing Alzheimer's disease (AD) pathophysiology, always by using sophisticated instrumentation for accurate detection. Here, we introduce CRISPR-AD, a CRISPR/Cas-based digital assay designed for the combined detection of protein and microRNA in blood. This method achieves a limit of detection (LOD) as low as 60 fg/mL for phosphorylated tau217 (p-tau217) and 0.5 fM for microRNA-34a-5p (miRNA34a), enabling successful detection in both AD patients and healthy individuals. We find that the combined use of these biomarkers improves the ability to distinguish between AD patients and healthy participants, particularly in individuals with mild cognitive impairment (MCI). Additionally, we have developed a portable device that integrates a smartphone as an imaging system for point-of-care testing (POCT), offering the potential for early stage AD screening. This study represents the first effort to evaluate the combined detection of blood protein and microRNA biomarkers for AD, underscoring the potential of multiple biomarker combinations for more accurate AD diagnosis.