Novel room-temperature RNA test achieves 10,000-fold sensitivity boost
Researchers at the University of Connecticut have developed CARRD, a novel RNA detection method that operates at room temperature without pre-amplification, achieving attomolar sensitivity for viral diagnostics. The technique detected HIV and hepatitis C virus RNA at concentrations 10,000 times lower than standard CRISPR tests, potentially transforming point-of-care diagnostics in resource-limited settings.
Standard CRISPR/Cas13a systems have revolutionised nucleic acid detection, but their reliance on pre-amplification steps and elevated temperatures has limited field deployment. Now, a team led by Professor Changchun Liu at UConn Health has eliminated these constraints through an ingenious exploitation of the Cas13a enzyme’s own regulatory mechanisms.
Exploiting molecular inhibition for signal amplification
The CARRD (CRISPR Anti-tag Mediated Room-Temperature RNA Detection) assay centres on a specially designed “CRISPR anti-tag hairpin” structure. This molecular mediator contains an eight-nucleotide anti-tag sequence that normally prevents Cas13a activation. When target RNA is present, however, the enzyme’s trans-cleavage activity cuts the anti-tag loop, exposing the target sequence within the hairpin and triggering a cascade amplification reaction.
“The target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a,” the authors explain in their Nature Communications paper (published online 15 October 2025). “By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection using a single CRISPR/Cas13a enzyme.”
The team optimised various hairpin designs, testing different lengths of complementary DNA blockers. A 14-base pair blocker proved optimal, effectively preventing premature activation whilst allowing efficient recognition after loop cleavage.
Unexpected temperature advantage
Surprisingly, the researchers discovered that Cas13a performs better at 25°C than the conventional 37°C. This finding contradicts earlier assumptions and significantly simplifies equipment requirements for field diagnostics. Testing across HIV and hepatitis C virus detection demonstrated the method’s versatility. “With CARRD, we were able to detect extremely small amounts of HIV and HCV RNA – down to 10 attomolar, which is 10,000 times more sensitive than standard CRISPR/Cas13a tests,” says Liu.
Clinical validation demonstrates diagnostic potential
The team validated CARRD using 30 deidentified clinical plasma samples. RNA extracted from 140 microlitres of plasma was tested without pre-amplification. Compared with RT-qPCR as the reference standard, CARRD achieved 77.8% sensitivity, 100% specificity, and 93.3% overall accuracy. The receiver operating characteristic curve yielded an area under the curve of 0.9709, indicating excellent diagnostic performance.
Two samples showed discordant results between CARRD and RT-qPCR, suggesting the technique may require further optimisation for samples with very low viral loads. Nevertheless, the positive predictive value reached 100%, with a negative predictive value of 91.3%.
Single-enzyme cascade amplification
Unlike competing approaches requiring multiple CRISPR enzymes – such as Cas13-Cas12 or Cas13-Csm6 tandem systems – CARRD achieves cascade amplification with a single Cas13a/crRNA complex. This dramatically reduces costs and simplifies the assay. The authors note that “using multiple Cas enzymes introduces challenges related to optimisation, scalability, and buffer compatibility, as well as increased costs and processing time.” The simplicity extends to readout as well. Fluorescent reporter cleavage provides a straightforward signal that could be adapted to paper-based detection or portable fluorescence readers.
Pathway to field deployment
“We successfully tested the method on real patient samples, detecting HIV RNA from clinical plasma, which shows the potential of this approach for low-cost, accurate viral testing in resource-limited settings,” Liu explains.
Future development will focus on alternative readout formats suitable for point-of-care use. The researchers acknowledge that hairpin design significantly influences background noise levels, and further optimisation could enhance specificity and reliability.
The method’s ability to operate at room temperature without pre-amplification or heating equipment addresses key barriers to deploying molecular diagnostics in low-resource environments. “Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools,” the authors conclude.
UConn has filed a patent application based on the technology, supported by NIH grants R33AI154642 and R01AI194917.
Reference: Moon, J., Zhang, J., Guan, X., et. al. (2025). CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a. Nature Communications, 16, 9142. https://doi.org/10.1038/s41467-025-64205-4
