Novel platform has potential to detect many disease-related biomarkers in just one test

Overview

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• Source: University of Würzburg


• Date: 28 Apr,2021

Most conventional molecular diagnostics usually detect only one disease-related biomarker. Fantastic examples are the PCR tests now used to diagnose COVID-19 by detecting a particular sequence from SARS-CoV-2.

Such so-called singleplex methods give reliable results because they are”calibrated” to a single biomarker. However, determining whether a patient is infected with a new SARS-CoV-2 version or a completely different pathogen requires probing for many different biomarkers at once.

Scientists from the Helmholtz Institute for RNA-based Infection Research (HIRI) and the Julius Maximilians University (JMU) in Würzburg have now paved the way for a completely new diagnostic system with LEOPARD. It’s a CRISPR-based method that’s highly multiplexable, with the potential to detect an assortment of disease-related biomarkers in just 1 test.

How LEOPARD works
This link allows LEOPARD to detect several RNAs simultaneously, opening opportunities for the simultaneous detection of RNAs from viruses and other pathogens in an individual sample.

The study published today in”Science” was pioneered by Chase Beisel, professor at JMU and research group leader in HIRI, and Professor Cynthia Sharma from JMU’s Institute of Molecular Infection Biology (IMIB). “With LEOPARD, we succeeded in detecting RNA fragments from nine unique viruses,’ says Beisel. “We were also able to distinguish SARS-CoV-2 and one of its variants in a patient sample whilst confirming that each sample was correctly collected from the individual.”

Background
CRISPR-Cas9 is principally known as a biomolecular tool for genome editing. Here, CRISPR-Cas9 be the molecular scissors that cut particular DNA sequences. These same scissors are naturally used by bacteria to cut DNA related to invading viruses.

Whether editing genomes or eliminating viruses, Cas9 cutting is directed by guide RNAs. The guide RNAs found in bacteria must pair with a separate RNA known as the tracrRNA.

An unexpected discovery
The tracrRNA was thought to only pair with guide RNAs coming from the antiviral system. However, the Würzburg scientists discovered the tracrRNA was pairing with other RNAs, turning them into direct RNAs.

When we searched for RNAs binding to Cas9 in our model organism Campylobacter, we surprisingly found that we detected not only guide RNAs, but also other RNA fragments in the cell that looked like guide RNAs. The tracrRNA was pairing with these RNAs, resulting in “non-canonical” guide RNAs that could direct DNA cutting by Cas9.”

Cynthia Sharma, Chair, Molecular Infection Biology II, Institute of Molecular Infection Biology

Sharma is also a spokesperson of the Research Center for Infection Diseases (ZINF) at JMU. The LEOPARD diagnostic platform builds with this discovery. “We figured out how to reprogram the tracrRNAs to decide which RNAs become guide RNAs,” says Beisel.

“By monitoring a set of matching DNAs, we can determine which RNAs were present in a sample based on which DNAs get cut. As part of this ongoing pandemic, LEOPARD could enable a doctor to figure out if the patient is infected with SARS-CoV-2, if it’s a special variant, and whether the sample was properly taken or needs to be replicated –all in one test.”

Later on, LEOPARD’s performance could dwarf even multiplexed PCR tests and other methods.

“The work highlights the fantastic collaborative and interdisciplinary research taking place here in Würzburg,” says Jörg Vogel, manager of IMIB and HIRI, a joint centre of JMU with the Helmholtz Center for Infection Research in Braunschweig. “LEOPARD impressively demonstrates that we are able to cover the complete spectrum of complementary cutting-edge study in Würzburg, from the essentials of RNA research to clinical applications”