The new method enables RNA detection with Cas12 nucleases

CRISPR-Cas systems, defense systems in bacteria, have become an abundant source of technologies for molecular diagnostics. Researchers at the Helmholtz Institute for RNA-Based Infection Research (HIRI) in Würzburg expanded this extensive toolbox. Their new method, called PUMA, enables the detection of RNA with Cas12 nucleases, which naturally target DNA. PUMA promises a wide range of applications and high precision. The team published their results in the journal Nature Communications.

Bacteria have developed special defense mechanisms to protect themselves from viruses, which by no means only infect humans. As part of these so-called CRISPR-Cas systems, a CRISPR ribonucleic acid (crRNA), which serves as a “guide RNA”, recognizes regions of a foreign genome, such as viral DNA.

The CRISPR-associated nuclease (Cas), directed by a crRNA, then renders it harmless by cutting it like a pair of scissors. Humans have taken advantage of this strategy: “CRISPR, often referred to as ‘gene scissors,’ is the basis of many molecular technologies,” says Chase Beisel, head of the RNA Synthetic Biology Division at the Helmholtz Institute for RNA-Based Infection Research (HIRI). ) in Würzburg. The institute is a site of the Braunschweig Helmholtz Center for Infection Research (HZI) in collaboration with the Julius-Maximilians-Universität (JMU) Würzburg, where Beisel holds a professorship.

The LEOPARD diagnostic platform, developed by Beisel’s lab in collaboration with JMU in 2021, also leverages CRISPR as a technology. LEOPARD has the potential to detect a variety of disease-related biomarkers in a single test. The approach is based on the reprogramming of RNA factors, so-called tracrRNAs. These RNAs are naturally involved in helping to produce guide RNAs used by Cas9 and by different Cas12 nucleases.

LEOPARD focused on Cas9. However, CRISPR-Cas systems also include another diverse set of nucleases, called Cas12.”

Chase Beisel, head of the RNA Synthetic Biology Division at the Helmholtz Institute for RNA-Based Research on Infection (HIRI), Würzburg

While both Cas9 and Cas12 cut DNA targets, Cas12 can increase the output signal by making cuts in “flanking” DNA. This can make detection technologies more sensitive and thus more effective.

The team led by Chase Beisel has now extended LEOPARD’s unique features to Cas12. The researchers named the resulting method PUMA (Pirogrammable tracrRNAs Unlock protospacer-neighbor Motif-independent ribonucleic acid detection ONEcids by Cas12 nucleases). Details of their findings are published in the journal Nature communications.

Overcoming obstacles

Although Cas12 nucleases are widely used in molecular diagnostics, two major limitations remain: Cas12-based technologies have been limited to DNA targets and a specific recognition sequence called PAM, short for protospace-neighbor patternrequired for recognition of the target molecule.

PUMA meets these challenges elegantly. Like LEOPARD, this new method is also based on tracrRNA. “Using PUMA, we can reprogram the tracrRNAs. This allows us to decide which RNA biomarker will become a guide RNA. This guide RNA, in turn, directs Cas12 to a DNA molecule we provide and activates the gene scissor,” explains the first of the study. author, Chunlei Jiao. Chunlei Jiao, a former graduate student and postdoctoral researcher in the Beisel lab, also participated in the development of LEOPARD. He recently started a professorship at the National University of Singapore. “The DNA cut then tells us which biomarker was present in the sample, such as biomarkers specific for different pathogens,” Beisel adds.

The new method therefore enables the detection of RNA biomarkers using CRISPR nucleases that can normally only recognize DNA. “This is particularly important for molecular biomarkers that can only be found at the RNA level. This includes RNA viruses, for example,” says Beisel. And yet, PUMA does not require a specific recognition sequence: PAM is contained in the DNA target molecule provided. Since researchers provide the target molecule, they can also insert cut DNA. As a result, they managed to significantly increase the speed of the method.

Many birds, one stone

“PUMA has the potential to become a versatile and precise tool for RNA detection,” concludes Beisel. Finally, the team demonstrated the potential of the method by identifying five bacterial pathogens associated with acute sepsis. Their detection was based on a single universal, reprogrammed tracrRNA, which provides a simplified means of differentiating between different types of bacteria. This opens up a wide range of potential applications in medicine: “The new technology represents a new form of CRISPR diagnostics that enables reliable molecular testing at the point of care—whether to identify viral or bacterial pathogens or to detect cancer biomarkers,” says Jiao.

The research team is already planning its next steps: “Our goal is to achieve a multiple readout similar to that of LEOPARD and expand the range of applications of the technology,” says Beisel, who also foresees widespread use in the research community: We hope that our study will provide impetus for further exploration of tracrRNA reprogramming.”