Cas12a2 enabled highly sensitive amplification-free RNA detection for SARS-CoV-2 in optimized assay conditions.
Evidence
This molecular diagnostic platform study optimized Cas12a2 biochemistry, reported a 46.7 aM viral RNA , tested 26 SARS-CoV-2 variants, and evaluated 317 clinical samples.
Caveat
The clinical validation described is limited to SARS-CoV-2 samples, so broader point-of-care performance across pathogens and settings is still inferred.
Simplified
Advances in clustered regularly interspaced short palindromic repeat () technologies have led to the use of diverse CRISPR-associated (Cas) proteins in diagnostic applications. Herein, we present a CRISPR/Cas12a2-based amplification-free RNA detection method that exhibits sub-attomolar sensitivity and substrate versatility. Cas12a2, a recently characterized RNA-guided nuclease, uniquely integrates bimolecular recognition through CRISPR RNA (crRNA)-target complementarity and protospacer flanking sequence identification, enabling highly specific trans-cleavage of single-stranded DNA, double-stranded DNA, and RNA. We have optimized key biochemical parameters, including pH, ionic strength, and temperature, to enhance the catalytic efficiency of Cas12a2. Based on the optimal activity conditions of Cas12a2, we have achieved ultra-sensitive viral RNA detection with a of 46.7 aM through the strategic design and cooperative activation of crRNAs targeting conserved regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The diagnostic accuracy of the Cas12a2-based assay has been demonstrated for 26 SARS-CoV-2 variants, and it has further resulted in the definitive diagnosis of 317 clinical samples. This work establishes Cas12a2 as a promising molecular diagnostic tool that provides an amplification-free, rapid, and versatile solution for RNA detection. The adaptability and simplicity of the platform render it particularly well suited for point-of-care applications, paving the way for next-generation CRISPR diagnostics.
Key numbers
46.7 aM
Achieved with the -based assay for SARS-CoV-2.
158 of 245 samples
Clinical Diagnosis Accuracy
100% sensitivity and specificity in clinical evaluations.
1000×
Sensitivity Improvement
Compared to single approaches.
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