Researchers have developed a new method called PUMA to detect RNA using Cas12 nucleases, expanding the capabilities of CRISPR-Cas systems. This technology promises high accuracy and a wide range of applications.
Bacteria have defense systems called CRISPR-Cas systems that protect them against viruses. These systems use a guide RNA (crRNA) and a Cas nuclease to identify and cut foreign DNA, rendering it harmless. Humans have harnessed this strategy for molecular technologies like CRISPR gene editing. The Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg has developed PUMA to detect RNA using Cas12 nucleases, building on their previous work.
One of the challenges in molecular diagnostics has been the limited use of Cas12 nucleases for DNA targets and the need for a specific recognition sequence called a PAM. PUMA overcomes these limitations by reprogramming tracrRNAs to guide Cas12 to DNA targets, allowing for RNA biomarker detection without a specific recognition sequence.
By reprogramming tracrRNAs, researchers can target RNA biomarkers, including those unique to RNA viruses, without the need for a specific recognition sequence. This flexibility improves the speed and accuracy of RNA detection using CRISPR technology.
The team demonstrated the effectiveness of PUMA by identifying bacterial pathogens associated with acute sepsis using a single, reprogrammed tracrRNA. This simplified approach can differentiate between different types of bacteria and has potential applications in molecular testing for various pathogens and cancer biomarkers.
Future plans for the technology include achieving a multiplexed readout and expanding its applications in research and clinical settings. The researchers hope that PUMA will encourage further exploration of tracrRNA reprogramming and enhance the capabilities of CRISPR-based diagnostics.
