Objective:

To develop a modified CRISPR–Cas12a system using DNA guides for RNA detection and cutting.

Key Findings:

• The DNA-guided Cas12a system accurately recognizes and cleaves RNA with strong sequence specificity.
• The system can detect RNA targets at low concentrations without preamplification.
• The assay showed complete agreement with RT-qPCR results in testing with SARS-CoV-2 clinical samples.
• The system can distinguish single-nucleotide sequence differences, aiding mutation detection.
• Minimal off-target effects were observed in transcriptome analysis.

Interpretation:

The study presents a new platform for RNA detection that could enhance existing CRISPR-based diagnostics.

Limitations:

• DNA-guided complexes were less stable than conventional RNA-guided systems.
• Intracellular RNA knockdown was modest.

Conclusion:

The findings indicate that programmable RNA targeting is achievable with DNA-guided CRISPR technology, suggesting potential for future molecular diagnostics.