FOCUS: A Dual-Mismatch crRNA Strategy Unlocks High-Fidelity One-Step SNV Detection with Cas12a

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR/Cas12a is a nucleic acid detection tool with limitations in single-nucleotide variant (SNV) discrimination.
• Existing methods require pre-processed templates and multi-step workflows, hindering clinical utility.
• Accurate SNV genotyping is crucial for diagnosing genetic disorders like spinal muscular atrophy (SMA).

Purpose of the Study:

• To enhance the single-nucleotide variant (SNV) discrimination capability of CRISPR/Cas12a.
• To develop a fast, one-step, universalizable CRISPR-based SNV detection system (FOCUS).
• To enable isothermal detection of SNVs directly from double-stranded DNA (dsDNA) for point-of-care testing (POCT).

Main Methods:

• Engineered crRNAs with site-specific dual mismatches to improve Cas12a SNV discrimination.
• Developed the FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system) platform.
• Integrated nucleic acid amplification and detection into a single isothermal reaction.
• Validated FOCUS using lateral flow strips and UV visualization for equipment-free POCT.

Main Results:

• Achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing SMN1 and SMN2 genes.
• Demonstrated 100% diagnostic concordance with gold-standard methods across 175 clinical samples for SMA, HPV, Staphylococcus aureus, and SARS-CoV-2.
• Successfully adapted FOCUS for low-cost, equipment-free lateral flow strip assays.

Conclusions:

• Established a generalizable engineering strategy for Cas12a crRNAs to enhance SNV detection.
• Presented FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping.
• Highlighted the translational medicine value of FOCUS for molecular diagnostics and point-of-care testing.