Page 14 - Sequence 2020-22

miSHERLOCK: CRISPR-based test to detect COVID variants in saliva

The device produces a fluorescent signal in response to the presence of SARS-CoV-2 viral RNA in a patient’s
saliva.

A simple, inexpensive CRISPR-based diagnostic test was recently created, that allows users to test themselves
for multiple variants of the SARS-CoV-2 virus at home, using just a sample of their saliva. Developed by
researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University, the
Massachusetts Institute of Technology, and several Boston-area hospitals, is a diagnostic device, called
Minimally Instrumented SHERLOCK (miSHERLOCK), is easy to use and provides results that can be read
and verified by a smartphone app within one hour.

miSHERLOCK successfully distinguished between three different variants of SARS-CoV-2 in experiments,
and can be rapidly reconfigured to detect additional variants like delta. It eliminates the need to transport
patient samples to a 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.

Simple things that used to be ubiquitous in the hospital, like nasopharyngeal swabs, were suddenly hard to
get, so routine sample processing procedures were disrupted, which is a big problem in a pandemic setting.
The team’s motivation for this project was to eliminate these bottlenecks and provide accurate diagnostics for
COVID-19 with less reliance on global supply chains, and also accurately detect the variants that were starting
to emerge.

For the SARS-CoV-2 detection diagnostic, the group turned to a CRISPR-based technology created in the lab
of Wyss Core Faculty member and senior paper author Jim Collins, called “specific high sensitivity enzymatic
reporter unlocking” (SHERLOCK). SHERLOCK makes use of CRISPR’s “molecular scissors” to snip DNA or
RNA at specific locations, with an added bonus: this specific type of scissors also cuts other pieces of DNA in
the surrounding area, allowing it to be engineered with nucleic acid probe molecules to produce a signal

indicating that the target has been successfully cut.

The researchers created a SHERLOCK reaction designed to cut SARS-CoV-2 RNA at a specific region of a
gene called Nucleoprotein that is conserved across multiple variants of the virus. When the molecular
scissors - an enzyme called Cas12a, successfully binds to and cuts the nucleoprotein gene, single-stranded
DNA probes are also cut, producing a fluorescent signal. They also created additional SHERLOCK assays
designed to target a panel of viral mutations in Spike protein sequences that represent three SARS-CoV-2
genetic variants: Alpha, Beta, and Gamma.

The team next focused their efforts on solving what is arguably the most difficult challenge in diagnostics:
Sample preparation.
7