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Study Demonstrates Multiplexing of Amplification-Free CRISPR MDx Technique

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NEW YORK – Using a technique called CRISPR-Cascade, a research team at the University of Illinois at Urbana-Champaign has developed a method to perform amplification-free molecular testing for multiple targets simultaneously. The technology, which was described on Monday in the journal PNAS, is being commercialized by startup VedaBio.

The proof-of-concept study was led by Rashid Bashir, a bioengineer who is the dean of UIUC's college of engineering and a cofounder of San Diego-based VedaBio.

In the study, the UIUC researchers showed that they could use their method to detect attomolar concentrations of target molecules. They also developed an OR gate using their chemistries to enable higher plexing and prototyped assays to detect four common bacterial causes of sepsis in contrived blood samples within 10 minutes.

As part of the bacterial adaptive immune system, clustered regularly interspaced short palindromic repeats, or CRISPR, proteins interact with CRISPR-associated, or Cas, proteins to identify and cleave non-host nucleic acids. While extremely accurate in this original context, when used in the lab, the approach typically requires nucleic acid amplification to increase sensitivity and specificity.

Bashir and his colleagues, however, have discovered that using CRISPR-Cas ribonucleoprotein complexes, or RNPs, can obviate the nucleic acid amplification step, essentially by creating a positive feedback loop in the reaction to generate exponential amplification of the reporter signal.

Specifically, they used an RNP containing a guide nucleic acid and a Cas enzyme specific for targets, such that binding with the target sequence initiates nucleic acid cleavage and also activates a fluorescent reporter. They also add a second RNP complex and a complex they called a blocked nucleic acid complimentary to the target sequence that is in turn cleaved by the first RNP, which together created an exponential reaction to produce a robust signal extremely rapidly.

The CRISPR-Cascade-based approach has a number of benefits, Bashir said in an email.

Namely, it serves to pool all the different targets into a single reaction, he said, with the OR-gate function — which produces a signal when any of the targets in the panel is present — enabling the reaction to signal the presence of targets in a binary way, thus "massively increasing" the potential coverage for multiplexing. The timescale of the reaction also makes it potentially useful for point-of-care testing.

"This publication formed the original thesis of the foundation of VedaBio," said Bashir. VedaBio was founded in 2021 and emerged from stealth mode in 2023. Now, "the company is focused on developing a robust platform meeting the highest standards of product development for clinical diagnostic applications," Bashir said.

Amplification-free molecular diagnostics have the advantage of being more rapid, and Bashir said that to his knowledge, VedaBio is the only company developing amplification-free CRISPR approaches.

Still, there is potential competition for VedaBio and the CRISPR-Cascade method among other CRISPR-based molecular diagnostics on the horizon.

Sherlock Biosciences, a startup that was acquired in December for $25 million by OraSure Technologies, has used isothermal amplification in its work, including a room-temperature approach. Meanwhile, Intelligenome is commercializing a CRISPR-based approach to detecting tuberculosis in blood samples that recently received breakthrough device designation from the US Food and Drug Administration.

In the academic realm, a team at the University of Florida is currently developing a CRISPR approach to rapid HIV self-testing, while a team at the Broad Institute, Harvard, and Princeton is working on paper-based CRISPR influenza diagnostics.

Sherlock previously obtained Emergency Use Authorization for a lab-based COVID test using its chemistries, as did Mammoth Biosciences, but there haven't been any cleared CRISPR diagnostic tests since then. OraSure said recently that it expects the Sherlock team to submit a marketing application with the FDA later this year for a 30-minute over-the-counter assay for chlamydia and gonorrhea.

Bashir also noted that the VedaBio team intends to make the CRISPR-Cascade technology commercially available. According to the study, the method could potentially be enhanced using a blood-drying technique the team has developed to improve the sample preparation step.