Enhanced SNV Detection of HLA-B*15:02TA by Integrating Blocking RPA and Inhibiting CRISPR-Cas12a

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas12a diagnostics (CRISPR-Dx) offer rapid detection of single-nucleotide variants (SNVs).
• Existing methods face challenges with complex procedures and suboptimal discrimination factors (DF), limiting their clinical utility.
• Previous approaches using RPA-based CRISPR-Cas12a require optimization for simplicity and enhanced specificity.

Purpose of the Study:

• To develop a simplified, highly specific, and sensitive one-tube assay for SNV detection.
• To improve the discrimination factor (DF) for distinguishing between wild-type (WT) and mutant targets (MT).
• To validate the assay's performance using a clinically relevant SNV associated with adverse drug reactions.

Main Methods:

• Proposed a one-tube "Blocking RPA-coupled Inhibiting CRISPR-Cas12a" (BRIC) cascade strategy.
• Implemented dual recognition of SNVs through inhibited Cas12a cleavage of WT targets and efficient MT detection.
• Utilized a 3D-printed portable fluorimeter for real-time detection and analysis.

Main Results:

• Achieved a significantly enhanced DF of 32.51 for SNV detection, with WT signal near background levels.
• Demonstrated a high sensitivity with a detection limit of 67.6 aM for the target SNV (HLA-B*15:02TA).
• Showed high consistency with next-generation sequencing in analyzing 22 clinical plasma samples.

Conclusions:

• The BRIC strategy offers a robust and simplified approach for SNV detection with improved specificity and sensitivity.
• The assay demonstrates significant potential for clinical applications, particularly in pharmacogenomics and personalized medicine.
• The developed method overcomes limitations of previous CRISPR-Dx techniques, paving the way for more accessible genetic diagnostics.