Enzymatic crRNA stabilization strategy for enhanced CRISPR/Cas12a detection.

The CRISPR/Cas12a system enables precise and rapid nucleic acid recognition through the programmable targeting capability of CRISPR RNA (crRNA). However, the intrinsic instability of crRNA limits the robustness and sensitivity of CRISPR-based molecular diagnosis in practical applications. Herein, we present a simple and general enhancement strategy that suppresses RNase-mediated crRNA degradation. This strategy, termed RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR), leverages RI to specifically prevent crRNA degradation, thereby improving its stability and enhancing the detection performance and anti-interference capability of the CRISPR system. Using influenza A virus (IAV) and mycobacterium tuberculosis (MTB) as model targets, RI-CRISPR improves detection sensitivity by nearly twentyfold compared to conventional CRISPR/Cas12a. Clinical validation using 40 MTB samples, combined with recombinase polymerase amplification (RPA), achieves 100% specificity and 96% sensitivity compared with the GeneXpert assay. Overall, this work provides a practical strategy to enhance sensitivity and robustness of CRISPR-based diagnostics, and is expected to promote further biomedical applications of CRISPR technology.