Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

Summary

The CasTDR platform pairs a topologically constrained DNA ring with Cas13a to accelerate post-amplification detection, achieving 0.1 aM sensitivity and single-nucleotide variant discrimination within 30 minutes. Engineered crRNAs with synthetic mismatches and hairpins enable robust differentiation of SARS-CoV-2 variants, demonstrated on clinical samples.

Key Findings

• Engineered topologically constrained DNA rings accelerate Cas13a trans-cleavage, enabling rapid post-isothermal amplification readout.
• Achieved 0.1 aM analytical sensitivity and single-nucleotide specificity in 30-minute sample-to-answer workflows.
• crRNA designs with synthetic mismatches and hairpin structures robustly discriminated SARS-CoV-2 variants in clinical samples.

Clinical Implications

Enables near-patient identification of viral variants to inform isolation, targeted therapies, and outbreak surveillance, especially where lab capacity is limited.

Limitations

• External multicenter validation and head-to-head comparisons with established NAATs are not reported.
• Operational performance in true point-of-care settings and varied sample types requires evaluation.

Future Directions

Prospective multicenter diagnostic accuracy studies, cost-effectiveness analyses, and extension to other RNA pathogens and resistance markers.