CRISPR-based COVID-19 diagnostic detects variants

A CRISPR-based diagnostic test has been designed that can be used at home to detect SARS-CoV-2 and multiple variants from saliva within one hour.

The self-contained diagnostic device is called minimally instrumented SHERLOCK (miSHERLOCK) and is the work of researchers at Harvard University and the Massachusetts Institute of Technology (MIT). SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) was first developed by a team led by Professor James Collins and Professor Feng Zhang at MIT in 2017 (see BioNews 897).

'miSHERLOCK eliminates the need to transport patient samples to a centralised testing location and greatly simplifies the sample preparation steps, giving patients and doctors a faster, more accurate picture of individual and community health, which is critical during an evolving pandemic, ' said co-first author Dr Helena de Puig, a postdoctoral fellow at the Wyss Institute and MIT.

This system uses RNA sequences to guide Cas enzymes to bind to specific RNA targets in the coronavirus genome. The Cas enzymes then cut at these specific RNA target sequences which generate a fluorescence signal in response to the presence of SARS-CoV-2 viral RNA in a patient's saliva. The team also designed SHERLOCK assays to detect three variants of SARS-CoV-2: Alpha, Beta and Gamma using a fluorescent signal also.

The diagnostic device, described in the scientific journal, Science Advances, can be built using a 3D printer and common electronic components for approximately USD $15, though researchers hope mass production could bring this down to around USD $3. Reuse could bring the cost per test down further. This system can also be accompanied by a smartphone app that was designed to analyse fluorescence and provide users with a clear 'positive' or 'negative' result. These results can also be shared with doctors and public health departments which facilitates easier tracking.

The battery-powered device requires the user to spit into the sample preparation chamber, transfer that sample into the reaction chamber and press a plunger to activate the SHERLOCK reaction. At the end of the reaction, the user can confirm the presence of a fluorescent signal by looking through the tinted transilluminator window or use the smartphone app to analyse the fluorescent readout.

When tested on clinical saliva samples from 27 COVID-19 patients and 21 healthy patients, miSHERLOCK was able to correctly identify 96 percent of COVID-19 positive patients and 95 percent of the healthy patients. They also tested the ability of the system to identify Alpha, Beta, and Gamma SARS-CoV-2 variants on artificial samples generated by spiking healthy saliva with synthetic viral RNA containing mutations present in each of the variants. The device was effective in identifying these variants across a range of concentrations.