Experimental technique promises to detect coronavirus in 40 minutes | Science



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American researchers are developing a rapid test for covid-19 that can diagnose an infection in 40 minutes. The new technology is still experimental and based on the revolutionary CRISPR gene editing technique.

This system works as a text editor that allows modifying the genetic material (DNA) of any living being. The technique was created in 2011 by the American Jennifer Doudna and the French Emmanuelle Charpentier and represented an unprecedented revolution in basic research. Little by little, it also opens the way in the experimental treatment of some diseases, including cancer. What a team at the University of California, San Francisco, using Doudna’s original idea, now demonstrates is that this powerful tool can also be applied to genetic material in new coronavirus, made of RNA.

“This technology is very interesting because it would allow us to develop a test that anyone could do at home very quickly,” says Jennifer Doudna, a researcher at the University of California, Berkeley. “This could reveal an infection, especially in asymptomatic cases.”

RNA is the intermediate molecule that reads instructions written in DNA and converts them into proteins, the molecules that perform vital functions. All living things, from microscopic bacteria to huge blue whales, need DNA to live and reproduce.

Coronavirus strategy is to infiltrate halfway through this process and convince our cells that it is not a stranger. This allows you to use cellular machinery to produce copies of yourself. Our genome is stored in a spiral formed by two very long strips of DNA that staple each other, like a zipper. The coronavirus material, in turn, is made from a single strip of RNA, and this is the weak point that the American team took advantage of.

CRISPR is inspired by the immune system of some microbes discovered by the Spanish Francis Mojica in 1993, when he studied single-celled beings from the salt flats of Santa Pola (east of the country). These organisms store fragments of the genome of the virus genome in their DNA that they find throughout their lives. When the virus reappears, the bacteria identifies it and activates a protein known as Cas, which cuts the virus genome in two and kills it.

We have a 50% chance of preparing this test for use in this pandemic

Jennifer Doudna, researcher at the University of California, Berkeley

What the San Francisco team did was create a CRISPR molecule that contains three elements: the genetic sequences of two genes for the new coronavirus, which serve as a guide; the Cas-12 protein, which serves as scissors; and a third fluorescent DNA fragment, published in the Biotechnology nature.

The exam uses a sample taken from the patient’s nose or mouth. First, chemicals are added to transcribe the RNA into the DNA, since Cas-12 only knows how to cut DNA. If there is a virus, the guiding part binds to the virus genome and activates Cas-12, programmed to cut strands of genetic material from a single strip in the sample. In this way, it also cuts the fluorescent strip and makes it glow. Finally, a lateral flow test is performed, a simple process, much like pregnancy tests, and then a second black bar appears indicating infection. The entire process takes just over 40 minutes.

This technology is based on the prototype developed by Doudna in 2018 and was patented by a company to which it is linked, Mammoth Biosciences. According to Doudna, the company requested the examination of the United States Food and Drug Administration (FDA) examination by emergency route, in the context of the covid-19 pandemicBut you still need to complete the detailed development to make the exam simple enough to take at home by following the basic instructions. “I think we have a 50% chance of preparing this test for use in this pandemic, if we can get through all the regulatory steps,” says Doudna.

Feng Zhang’s team, another pioneer of the CRISPR technique who works at the Broad Institute at MIT and Harvard and who is involved in a long legal battle against the University of California for patents on this technology, also developed a similar test for the new coronavirus, which in this case is able to directly detect the RNA of the virus without the need to transcribe it.

“I think we are seeing the beginning of a second conflict, since we are the first to apply this technique to the diagnosis of covid-19,” says Lluis Montoliu, a researcher at the National Center for Biotechnology (CNB) in Spain. “On the one hand, in the test, Doudna’s ability to detect the virus is one-tenth of the PCR. This is not a problem if the viral load is medium or high, but it could lead to false negatives if the person is very early in infection or coming out of it, “he explains. On the other hand, this technique requires simpler chemical reagents than PCR and does not need specialized personnel to perform it, which is an advantage ”, he details.

The next step in that race was evident, and the first to take it was another EE team. USA, Led by Lei Qi of Stanford University. His team developed a CRISPR to detect RNA from the virus and kill it by cutting it in half. The coronavirus genome is a single strip of RNA. At each end there are protective molecules that mask it so that it cannot be detected and removed. The new tool, called PACMAN (“Human Cell Prophylactic Antiviral CRISPR”), targets an intermediate region of the genome, with which it can seamlessly bind and activate the scissors, which in this case is the Cas-13d protein, capable of cutting RNA directly. The system is capable of eliminating 80% of the genetic sequences of the coronavirus in human lung cells, according to a study published in Biorxiv, even without review by other experts. The work adds that only six variants of CRISPR would be necessary to eliminate 90% of all known coronaviruses. This tool “has the potential to be rapidly implemented to combat pandemic variants of the coronavirus,” the authors of the paper write.

This type of genetic editing would be more feasible than the one focused on DNA, since it does not need to penetrate the nucleus of cells and manipulate their sacrosanct sequence, where any introduced fault or defect can cause damage or pass from generation to generation. But RNA is an essential molecule for life, and this type of intervention may cut not only the RNA of the virus, but also that of its host. The Cas-13d molecular scissors are more accurate in this regard, as they do not cut indiscriminately.

On the one hand, in the Doudna test, the virus detection capacity is one tenth of the PCR. On the other hand, this technique requires simpler chemical reagents than PCR and does not need specialized personnel to perform it, which is an advantage.

Molecular biologist Miguel Ángel Moreno Mateos, researcher at the Andalusian Center for Developmental Biology, is one of the leading Spanish specialists in these molecular scissors. In a recent study, his team explored the effectiveness of Cas-13d in zebrafish and rat embryos. “What we saw is that the technique is clean, safe and efficient, since it cuts 80% of the target strings and does not cause damage; animals develop normally, “he observes.

Later this month, a team of Chinese scientists announced the application of these scissors for the first time in adult animals. They have shown that they can administer CRISPR to mice suffering from neuronal death and transform the immune cells of their cerebral glia into new neurons, thus alleviating paralysis and Parkinson’s-like symptoms, as explained in the magazine. Mobile. The work points out that this may be a new way of treating neurological diseases without treatment in humans.

But, like any other possible therapy, these versions of CRISPR now face the so-called valley of death: the long process of preclinical validation in animals and the necessary tests in humans, in which more than 95% of all experimental treatments fail. .

“This is a promising system, but there is still a need to develop a way to transmit it safely and efficiently into the patient’s cells, for example to his lungs, without causing damage,” says Moreno Mateos, whose team uses this technology to disable specific RNAs for basic studies on the function of certain genes in animals. “As for therapies for covid-19, the most currently available are those that are already approved for other uses. The point is that neither of them is likely perfect, and this is where CRISPR has a lot to say about its ability to specifically degrade the genome of this and other viruses. There is a very promising field of research, “he says.

Information about coronavirus:

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– What to do to protect yourself? Questions and answers about coronavirus;

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