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Of all the The genome modification tool known as Crispr, which is being re-birthed, may not be more appealing than its potential to publish some of humanity's worst diseases. from the history books. This week again, Crispr Therapeutics announced that it has begun treating patients with beta-thalassemia, a hereditary blood disorder, during the first test of genetic disease technology in the drug industry. Despite the progress, there is still a host of unknowns that prevent the widespread use of Crispr-based medicines, including safety.
This is because the classic and most widely used version of Crispr works by cutting a strand of DNA at a specific place in the genome and letting the cell sew it together. The major concern is that an army of DNA-breaking enzymes can sometimes go astray and cause unexpected mutations in places it should not. When a more precise technique, called Basic Edition – which permutes individual letters of DNA without cutting the strand – arrived in 2017, it promised a safer path. The technique had a specific potential for two-thirds of the 50,000 or so human genetic diseases caused by a single letter, and investors have not wasted time getting a license for the technology. Researchers in China immediately began testing such a basic editor on viable human embryos.
It now seems like it was premature. Using a new method of measuring unplanned changes, a team of American, Chinese and European scientists discovered that the same basic editor, widely used by today's researchers, is disrupting the genome at a breakneck pace.
Their report, published today in Science, claims a 20-fold increase in the number of mutations compared to what would be expected in the normal course of cell division and repair in mouse embryos "This number varies depending on many factors. However, if you want to move this basic editor into a clinical setting, you should probably worry, "says Stanford biochemist Lars Steinmetz, co-author of the paper. This advice is particularly interesting, he says, to scientists who might be tempted to circumvent the rules and regulations to impose on the editors the essential bases, which has been preoccupying him since the Crispr baby scandal in November.
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The WIRED guide of Crispr
David Liu, the biochemist whose lab at Harvard and the Broad Institute has developed the basic editor in question, is not as sure of the clinical impact. The Steinmetz group discovered nearly 300 additional mutations in edited cells, as opposed to unedited cells. Three hundred errors on the six billion bases of the mouse genome give a mutation rate of one in 20 million. Liu says this number is within the range of errors that your cells spontaneously commit themselves – more than neurons, but less than skin cells. Despite everything, he says Science Paper is an important step forward in a field that always sets its standards for safety. "It's an intelligent and elegant method designed to amplify the signal so that we can now detect and understand these rarer types of off-target events, independent of the guides."
A bit of background: in 2017, Nature Methods published a one-page letter claiming that a Crispr treatment that cured two blind mice had also caused a large number of involuntary mutations. Crispr stocks dropped and scientists cast a shadow over the dramatic results, which were based on sequencing each mouse and comparing their DNA to unedited siblings. The paper was finally removed and the changes were determined to be simply the natural genetic variation between different individuals of the same laboratory strain. But the episode has highlighted an important blind spot in these error detection technologies. Virtually all use a kind of algorithm to choose locations in the genome where Crispr is likely to accidentally go to work and observe what happened there. "This is an area in which you only see what you are looking for," says David Jay Segal, a molecular geneticist who studies these effects at the UC Davis Genomics Center and did not participate in any of these studies. Scanning the entire genome for changes would be ideal, he says. But nobody had found a way to do it in live animals with the appropriate controls up to Steinmetz's group.
The trick was to make each animal sound own control. Stay with me. Scientists used mouse embryos that had only one age of cell division, two cells in total. In one cell, they injected Crispr editing constructs and a reporter protein called tdTomato. The other cell, they left alone. And then, they let the embryos grow for 14 days. Under a special type of microscope, all the modified cells were bright red, whereas all unmodified cells remained dark. They used this fluorescence to sort the cells, sequence them and compare the six billion base pairs.
The idea was to design a method that could detect any unintended change for any type of Crispr system, Steinmetz explains. "We wanted to be able to get modifications through mechanisms that we do not understand yet." Consider it as a red flare gun: it can send a warning signal, even if no one knows exactly what that is happening.
Well, not really anyone. The authors assume that one of Liu's editors tends to grab the bare, single-stranded DNA he discovers. In a fast-dividing embryo, many such DNAs are exposed to cells, allowing the basic editor to mess up. Liu, who knows more than anyone else, says it's perfectly fine.
Because this particular publisher can so well bind DNA alone, even without its Crispr guide, "it's no wonder to see these unwanted issues." Liu, whose core editors have been licensed by an $ 87 million start-up that he co-founded, says his lab has already developed a number of more specific versions of his original basic editing approach . This work is still in preparation for publication, he says.
"There is no doubt in my mind that this will be a quick fix," says Steve Murray, principal investigator at the Jackson Laboratory in Bar Harbor, Maine. It is part of a consortium of 17 academic research institutes that have received $ 190 million from the National Institutes of Health over the next six years to develop safeguards and standards for genome editing for therapeutic purposes. He says that there is actually a bigger story with the new Science paper. In addition to the core editors, the Steinmetz group also tested the software 'Ol Crispr 1.0, the gene editor of the world of biological research. And they found that the test was successful. In Murray's mind, this is the first time that convincing evidence shows that Crispr Classic is not the victim of any mechanically mysterious mistake. As long as you do a good job telling him where to go, he will do the job you have designed. "It is useful to settle this debate back on the untargeted targets that no previous study has actually been designed to answer properly."
Murray's question now is, how many mistakes are there? Cells are prone to making their own mistakes – in the order of once a million to 100 million base pairs, with more skin cells and fewer sperm and eggs. Is it important if an overactive gene editor makes this number closer to one in 500,000? What is a damaged gene in a cell on 37 000 billion? And if the mistake in this cell became a cancer? And if a patient is on the verge of death, how important is it?
Murray and his colleagues at the NIH consortium will spend the next six years solving some of these issues. He imagines that they will one day be able to calculate risk curves to help doctors and regulators assess the trade-offs between Crispr-based medicines. "I do not think that we or anyone can find an absolute rule, that beyond a certain number of errors, it is useful for nothing and below which it is useful for all. Every disease is different. Each treatment is different. Every patient is different, "says Murray. "But you must first have the data to start the discussion." Crispr therapies, it seems, must now pass an additional test.
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