CRISPR-based tests to speed up the diagnosis of COVID-19 – Québec Science

Ultra-sensitive tests, based on CRISPR technology, could hit the market in the coming months.

Their advantage? They give a response in 5 minutes, detect tiny amounts of viruses and work with a simple mobile phone.

It is Jennifer Doudna, one of the 2020 Nobel Prize winners in chemistry awarded for the development of the CRISPR-Cas9 molecular scissors, who proposes the technique in a pre-publication in collaboration with the team of Melanie Ott, from the University of California at San Francisco.

To understand how this test works, it should be remembered that CRISPR-Cas9 is a tool that allows you to precisely locate any DNA or RNA sequence, and to cut the genetic material at the location in question. . Used in laboratories around the world to modify genes, it can also be used to spot the coronavirus.

The principle is simple: CRISPR is used to locate a small genetic sequence specific to the SARS-CoV-2 coronavirus, then to cut said sequence. In doing so, the genetic scissors indirectly release a fluorescent compound, which reveals the presence of the virus.

Increase the number of researchers

As of last May, CRISPR tests developed by the American company Sherlock Biosciences were urgently approved by the Food and Drug Administration in the United States. In July, another American company, Mammoth (co-founded in 2017 by Jennifer Doudna), also obtained marketing authorization.

The two tests work in the same way: you must first take a nasopharyngeal sample, then amplify the genetic material of the virus before using the genetic “scissors”. The results are obtained in less than an hour and make it possible to do without expensive and bulky PCR machines. Other companies all over the world and particularly in India are developing similar inexpensive tests.

But the team of Melanie Ott and that of Jennifer Doudna and Daniel Fletcher at the University of California at Berkeley further refined the technique using a slightly modified tool, CRISPR-Cas13 (an analogue of Cas9), which has three “research heads” instead of one. These are in fact small RNAs, called “guide RNAs”, which allow the CRISPR tool to locate its target, and they can be modified at will.

By testing numerous trios of guide RNAs, the researchers significantly improved the sensitivity of the test. “We are using three guides simultaneously to detect SARS-CoV-2 virus RNA. This allows many Cas13 proteins to attach to different places on a single RNA strand, rather than having a single protein attached. This increases the sensitivity and also offers protection against possible mutations – if the virus were to mutate at one of the attachment sites, the other two targets would remain functional ”, explains to the study’s first author, Parinaz Fozouni.

This even makes it possible to “skip” the amplification step and directly detect the virus in the samples taken from the patients, after having extracted the RNA (which is already done for the other tests). “We are currently working on a one-step test that would not even require extracting the RNA from the swab,” adds the researcher.

By using a single “guide” to pinpoint the viral sequence, researchers are able to detect concentrations of 100,000 viruses per microliter of solution. With multiple guides, they manage to detect intruders even if there are as few as 100 per microliter, the magazine reports. Science.

This test in development has the advantage of quantifying the viral load, that is to say the quantity of virus present: all you need is a cell equipped to analyze the degree of fluorescence of the sample. “The phone can pick up a wide range of fluorescence, and the amount of fluorescence emitted is proportional to the viral load,” explains Parinaz Fozouni. Because we don’t amplify RNA like with other diagnostic tests, we keep the initial quantitative differences. ”

“This test can offer rapid, low-cost and portable screening,” note the authors, who want to bring it to market quickly.

Rapid tests approved in Canada

Tests based on CRISPR technology are not the only rapid diagnostic tests available. Health Canada has just approved a so-called “antigenic” test, provided by Abbott Rapid Diagnostics, which detects the presence of SARS-CoV-2 surface proteins using antibodies.

“Testing is a pillar of Canada’s response to the COVID-19 epidemic and that is why Health Canada has prioritized the evaluation of all types of COVID-19 tests. These antigenic tests will have a role to play in preventing major outbreaks, when rapid results are necessary to counter the spread of the virus, ”said Federal Minister of Health, Patty Hajdu.

Canada has also approved the ID Now test, used for several weeks in the United States, which detects the genetic material of the virus in less than 15 minutes, using a portable device that is lighter than conventional PCR machines.

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