The team at Case Western Reserve University in Cleveland, Ohio, harnessed CRISPR's precision to identify and quantify viruses in human samples, including serum from blood. Their technique, named E-CRISPR, could offer a robust, accurate and cost-effective way to enable faster diagnoses of infections such as parvovirus which can cause miscarriages and human papillomavirus (HPV) which is associated with some cancers.
'This could someday become a simple, accurate and cost-effective point-of-care device for identifying different nucleic acid viruses, such as HPV or parvo from a single droplet of a blood sample,' said first author Yifan Dai, a PhD candidate at Case Western. 'And it would also be extremely fast.'
HPV and parvovirus are both DNA viruses, and their genomic material will be present in the blood of an infected person. E-CRISPR uses a CRISPR RNA strand to bind target sequences which are unique to the virus, and when attached, the associated Cas12a (also known as Cpf1) enzyme cuts the viral DNA in the same way as it would in CRISPR/Cas9 genome editing.
However, unlike Cas9, Cas12a is known to indiscriminately cut single-stranded DNA (ssDNA) once activated by the double-stranded target – in this case the viral sequence. The researchers used ssDNA strands, tethered at one end to a sensor and with an electrochemical tag molecule at the other to detect if this cut has been made. If Cas12a is activated, these ssDNA strands are cut, detaching the tag molecules and the electrochemical current detectable through the sensor drops, giving an observable result.
'The CRISPR technique works so that it cuts all of the non-specified single-strand DNA around it once the target is recognised, so we program to electrochemically probe this activity,' said Dai. 'No virus – no cutting, it's that simple. And the opposite is true: If CRISPR starts to cut, we know the virus is present.'
The researchers hope that this method could be further developed to create a new 'universal biosensing' device able to accurately detect viruses quickly at the point-of-care, similar to existing blood-glucose sensors. Currently, systems that detect HPV and parvovirus take days to process.
The study was published in Angewandte Chemie, a journal of the German Chemical Society.