A novel CRISPR-powered diagnostic test for mpox

Researchers have developed a fast, specific and reliable test for mpox infection, combining CRISPR/Cas12a and nanopore sensing.

Research presented at the 68th Biophysical Society Annual Meeting (10-14 February 2024; Philadelphia, PN, USA) by Md. Ahasan Ahamed of Pennsylvania State University (PA, USA), reveals a recently developed, fast and accurate diagnostic test for mpox, formally known as monkeypox. This approach repurposed the bacterial CRISPR/Cas12a system and has the potential to replace the current standard polymerase chain reaction (PCR) tests for mpox, which are more time-consuming, resource-intensive and require specialist equipment. The same principle could also be applied to produce many other diagnostic tests for different viruses.

Mpox is caused by monkeypox virus (MPVX), which can spread through close contact. While those with mpox present typically with mild symptoms such as fever and rash, severe cases also occur, which require much more intensive medical intervention. As mpox is easily spread between people, producing rapid diagnostic tests to quickly identify those who need to isolate is important in developing strategies to prevent this spread, alongside other approaches such as vaccination.

Currently, diagnostic testing for mpox takes a few hours and relies on PCR to specifically amplify sections of genetic material from MPVX within samples. This also requires PCR machines capable of cycling through specific temperature points to ensure successful amplification. The researchers set out to develop an alternative to this PCR test and reduce the time before results are obtained.

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The newly developed approach repurposed CRISPR/CAS12a, an RNA-protein complex that forms part of an antiviral mechanism in bacteria. Reporter sequences were designed by the team to bind to MPVX genetic material, while also being bound by a  programmable CRISPR RNA. The associated CAS12a protein then cleaved these reporters into fragments. These fragments were then amplified using an alternative method to PCR that does not require thermal cycling, recombinase polymerase amplification, and detected using a nanopore sensor. Detection of these fragments indicated the initial binding of the reporter to MPVX and demonstrated the presence of the virus in the sample.

The described strategy produced reliable positive results in samples containing MPVX, while remaining highly specific, as samples containing the related cowpox virus did not produce positive results. Researchers are also keen to highlight the speed of this test compared to the existing approach: “In total it takes 32 to 55 minutes to detect the target, depending on viral load,” elaborates Ahamed, the lead author of this study.

While this is a huge step in producing a fast, specific and reliable rapid test for mpox, researchers also hope these findings can be applied to diagnostic tests for a variety of viruses. Amahed aims to be able to produce a device that could make this new modality of pathogenic testing accessible widely.