Publication date: Aug 09, 2026
CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13. 15 aM) and ultrafast readout (
| Concepts | Keywords |
|---|---|
| CRISPR/Cas12a | |
| dual-mismatch crRNA | |
| FOCUS | |
| high-fidelity | |
| one-pot assay | |
| single nucleotide variants |
Semantics
| Type | Source | Name |
|---|---|---|
| drug | DRUGBANK | Gold |
| disease | MESH | spinal muscular atrophy |