Researchers have developed a modified CRISPR–Cas12a system that uses DNA guides instead of the RNA guides typically required by CRISPR technology to detect and cut RNA targets. The findings may expand future options for molecular diagnostics and RNA-targeted therapies.
In the study, published in Nature Biotechnology, investigators engineered synthetic DNA guides that direct Cas12a to recognize specific RNA sequences. Unlike conventional CRISPR systems, which rely on RNA guides to locate genetic targets, the new approach separates the targeting and activation steps by using DNA as the guide and RNA solely as the target.
Laboratory experiments showed that the DNA-guided Cas12a system could accurately recognize and cleave single-stranded RNA while maintaining strong sequence specificity. The platform also generated “trans-cleavage” activity, a reaction commonly used in CRISPR-based diagnostic assays to produce detectable fluorescent signals.
Researchers reported that the system was able to detect RNA targets at very low concentrations, including without preamplification in some experiments. They also developed a diagnostic workflow called SLEUTH, which combined isothermal amplification with DNA-guided Cas12a detection. In testing with 31 SARS-CoV-2 clinical samples, the assay showed complete agreement with RT-qPCR results within the study cohort.
The DNA-guided system appeared to selectively target RNA rather than DNA. The investigators also found that the assay could distinguish single-nucleotide sequence differences, particularly within key target regions, which may support future applications in mutation detection and precision diagnostics.
The study also explored whether the system could function inside living cells. Using HEK293T cells, the researchers demonstrated targeted reduction of EGFP messenger RNA expression with chemically modified DNA guides. Transcriptome analysis suggested minimal off-target effects in the experimental model.
Overall, the study highlights a possible new platform for RNA detection that could complement existing CRISPR-based diagnostics. Because DNA guides are generally more stable and easier to manufacture than RNA guides, the approach may offer practical advantages for assay development, storage, and large-scale production.
The authors noted that the system still requires further optimization. The DNA-guided complexes were less stable than conventional RNA-guided CRISPR systems, and intracellular RNA knockdown remained modest. However, the findings demonstrate that programmable RNA targeting can be achieved using DNA-guided CRISPR technology, potentially broadening the future design of molecular diagnostic and RNA-targeting tools.
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