Viruses can mutate, edit, or shuffle their genetic material, meaning diagnostic tests may fail to catch them. The genetic sequence variations in new strains may impede the ability of some diagnostic tests to detect the virus.

A scientists team led by Nanyang Technological University, Singapore (NTU Singapore) has developed a diagnostic test that can detect the virus that causes COVID-19 even after it has gone through mutations.

They uses the CRISPR gene editing tool to make the diagnostic applications against virus. They call it VaNGuard (Variant Nucleotide Guard) test.

In addition to its ability to detect SARS-CoV-2 even when it mutates, the VaNGuard test can be used on crude patient samples in a clinical setting without the need for RNA purification, and yields results in 30 minutes. This is a third of the time required for the gold standard polymerase chain reaction (PCR) test, which requires purification of RNA in a lab facility.

The team present a CRISPR-based diagnostic assaythat is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, the use of an engineered CRISPR AsCas12a enzyme enables detection of wild type and mutated SARS-CoV-2 and allows to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65°C.

The VaNGuard test relies on a reaction mix containing CRISPR enAsCas12a protein, a variant of the enzyme CRISPR Cas12a that acts like a pair of "molecular scissors".

The CRISPR enzyme enAsCas12a is 'programmed' to target specific segments of the SARS-CoV-2 genetic material and to snip them off from the rest of its viral genome. Successfully snipping off segments is how the enzyme 'detects' the presence of the virus. The programming is done by two different molecules known as guide RNAs, gRNA, which are designed to recognise specific sites on the SARS-CoV-2 genome.

The scientists decided to use two guide RNAs that recognise sequences that are extremely similar between variants of SARS-CoV-2 and that are also unique to the virus. Each guide RNA is computationally predicted to recognize over 99.5 per cent of the thousands of SARS-CoV-2 isolates that have so far been sequenced around the world.

Combining two or more guide RNAs with the enzyme enAsCas12a ensures that if one of the guide RNAs fails to guide it to the correct segment of the virus because of amutation, the other guide RNA can still 'rescue' this mismatch.

So far, the made-in-NTU diagnostic platformcan recognize up to two mutations within the target sites on the SARS-CoV-2genome.

When the SARS-CoV-2 virus or one of its variants is detected in a sample, the engineered CRISPR Cas12 enzyme varianten As Cas12a becomes hyper-activated and starts cutting other detectable geneticmaterial in the sample as well, including a molecule tagged with a fluorescentdye that is added to the reaction mix.

When the molecule is cut, it starts toglow. This glow is picked up by a microplate reader, a lab instrument that can detect and quantify the light photons emitted by the molecule.

If the virus is present, the molecule will glow. If not, it means the virus is not present to cause the hyper-activation of the molecular scissors.

To make the test easier to use once it has been approved for roll out, the scientists integrated the test into a specially treated paper strip that looks similar to a pregnancy test.

The paper strip is dipped into a tube containing the crude nasopharyngeal sample and the reaction mix. In the presence of a SARS-CoV-2 virus or its variant, two strong bands will appear onthe paper strip. In the absence of the virus, only one band will appear.

The scientists validated the VaNGuardtest's ability to detect SARS-CoV-2 variants by synthesising an RNA sample that has the same mutated sequence as a known SARS-CoV-2 variant.

They added different amounts of the synthesised sample to their test and observed two strong bands when the paperstrip was dipped into each reaction mix. This indicates that the VaNGuard testis robust against mutated viral sequences. The scientists also developed amobile phone app to facilitate the interpretation of the paper strips.

The team also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling the test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, they demonstrate that the test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

reference:

An engineered CRISPR-Cas12a variant and DNA-RNAhybrid guides enable robust and rapid COVID-19 testing