Enzymatic crRNA stabilization strategy for enhanced CRISPR/Cas12a detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• CRISPR/Cas12a systems offer precise nucleic acid recognition via CRISPR RNA (crRNA).
• crRNA instability due to RNase degradation limits the sensitivity and robustness of CRISPR-based diagnostics.
• A need exists for strategies to enhance crRNA stability for improved diagnostic performance.

Purpose of the Study:

• To develop and validate a simple, general strategy to suppress RNase-mediated crRNA degradation.
• To enhance the stability, sensitivity, and anti-interference capability of CRISPR/Cas12a systems.
• To improve the practical applicability of CRISPR technology in molecular diagnostics.

Main Methods:

• Development of RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR) strategy.
• Utilizing RI to specifically prevent crRNA degradation.
• Testing RI-CRISPR performance using influenza A virus (IAV) and Mycobacterium tuberculosis (MTB) as model targets.
• Clinical validation of RI-CRISPR combined with recombinase polymerase amplification (RPA) for MTB detection.

Main Results:

• RI-CRISPR significantly enhances crRNA stability, improving detection performance and anti-interference capabilities.
• Detection sensitivity was improved nearly twentyfold compared to conventional CRISPR/Cas12a for IAV and MTB.
• Clinical validation with 40 MTB samples demonstrated 100% specificity and 96% sensitivity compared to the GeneXpert assay when combined with RPA.

Conclusions:

• RI-CRISPR is a practical and effective strategy for enhancing the sensitivity and robustness of CRISPR-based diagnostics.
• This approach addresses crRNA instability, a key limitation in CRISPR diagnostic applications.
• The enhanced CRISPR system shows significant potential for further biomedical applications, particularly in infectious disease diagnostics.