In search of an antidote to CRISPR
Is it possible to reverse changes in genes?
Ilya Khel, Hi-News.ru , based on Arizona State University: Proofreading the book of life: gene editing made safer
When the scientists behind the Manhattan Project learned about the destruction of Hiroshima and Nagasaki, enthusiastic enthusiasm was replaced by gloomy regret. What began as a revolution in the field of physics mutated into a weapon of mass destruction, from which it was impossible to defend. From the point of view of biology, CRISPR has a destructive power of similar proportions. And scientists don't want history to repeat itself again.
CRISPR: Nuclear weapons in the world of biology
Just five years after the discovery of CRISPR, the Defense Advanced Research Agency DARPA initiated the Safe Genes program: a collaboration of seven of the world's leading experts in the field of gene editing, aimed at finding a variety of antidotes to CRISPR and improving gene editing capabilities in space and time.
The point is not to fuel public fear of a powerful tool; rather, it is necessary to see potential dangers in advance and find preventive measures or countermeasures. If CRISPR is a biological Pandora's box, it is already open: human trials of CRISPR have begun in clinics; in the laboratory, the technology is turning into gene drives capable of destroying entire species. The goal of the Safe Genes program is to find a way, or many ways, to close this drawer again.
Last week, the search for the CRISPR antidote became even more active. A team led by Dr. Amit Choudhary of the Broad Institute at the Massachusetts Institute of Technology, a member of Safe Genes, has developed a "screening" platform for quickly sifting through more than 10,000 small chemicals that reduce the activity of Cas9 scissors.
The team refined the structure of several promising candidates to further enhance their ability to counteract CRISPR, and created two antidote molecules that prevent Cas9 from binding to the DNA target and cutting it. During tests on human cells in Petri dishes, the molecules floated through cell membranes and reliably destroyed CRISPR activity within a few minutes.
These drugs are the very first candidates – and they can be even more toxic than even CRISPR, which has gone wild inside the body. Scientists will have to test them on animals to assess their effectiveness and safety.
But small drugs that counteract CRISPR, even the very first ones, prove that CRISPR titanium can be stopped. With the screening platform, there is a chance to find even more powerful "cancel" buttons: chemicals that may one day turn into injections or pills that block unwanted gene editing activity, in the medical field, or even in biological weapons.
"These results lay the foundation for precise chemical control of CRISPR/Cas9 activity, paving the way for the safe use of such technologies," says Choudhary.
What is already available at the moment?
Choudhary is not the only one from the Safe Genes team looking for anti-CRISPR molecules.
In 2013, another member of the project, Dr. Joseph Bondi-Denomi from the University of California, San Francisco, helped find the first anti-CRISPR drugs: large, clumsy proteins that block the Cas9 scissors, preventing them from recognizing DNA molecules or contacting them. His brilliant idea was to return to the natural roots of CRISPR as a system of bacterial immune protection against viruses.
At its core, CRISPR allows bacteria to store a "criminal's photo card" – viral DNA – in their own genome, so that when the virus attacks again, the Cas scissors can tear the virus into pieces. But phages are not misses either. In the evolutionary race, they also developed genes that create anti-CRISPR proteins to counteract the immune defenses of bacteria.
Turning to the biology of phage anti-CRISPR in 2012, Bondi-Denomi discovered several new proteins that widely inhibit the activity of Cas12a, an alternative to Cas9, which is gaining popularity as a diagnostic tool. Working separately, Jennifer Dudna from the University of California, Berkeley, one of the first discoverers of CRISPR and also a member of the project, used bioinformatics to track down a handful of Cas12a killers that block gene editing activity in grown human cells.
At the time, Dudna said these results "pave a direct path to the discovery of even more anti-CRISPR from the microbe world."
Tiny lights
And yet, anti-CRISPR proteins are not particularly useful switches in the real world.
Proteins are difficult: they are large and clumsy, so they cannot penetrate into cells and bite into CRISPR mechanisms. They are sensitive to changes in temperature and digestion and do not live very long inside the body. Many of them become attractive prey for our immune system, which can trigger irritating – if not dangerous – allergic reactions.
Small molecules, as a rule, do not have such problems. They are fast, cheap and their effects are reversible. Don't want to interfere with CRISPR? Just wait for the molecules to wash out. But effective ones are very hard to find.
And this is where the Choudhary screening platform will help.
The high-performance system sifts through tens of thousands of chemicals using two tests, looking for promising ones. First, she monitors the DNA segments by binding to the Cas9 scissors with neon lights. DNA is marked with fluorescent "bulbs" that change polarization when bound to Cas9, similar to how polarizing glasses change in sunlight. This allows the team to quickly track whether the molecule breaks the Cas9 bond with DNA.
Secondly, the system uses automatic microscopes that look for fluorescent signals in cells acquired or lost during Cas9 activity. In one of the studies, scientists used cells that usually glow green until Cas9 cuts out the gene. A potential anti-CRISPR drug will allow cells to remain green even in the presence of a dose of CRISPR.
In the process, scientists have identified the BRD0539 molecule, which does not allow Cas9 to bind to the target DNA sequence. The effects of the drug were completely predictable: the higher the dose, the more it suppressed CRISPR activity.
These results are already helping to reduce the side effects of CRISPR in therapeutic settings. In cells, the dose of such a drug rapidly reduced the ability to cut Cas9 by half, which, in turn, reduced the unintentional cutting of the HBB gene involved in sickle cell anemia by five times.
It's not hard to imagine a future in which you can take a pill from BRD0539–or a more powerful next–generation equivalent-to temporarily reduce or stop the effects of CRISPR before it goes rogue in your body. The drug, being a small molecule, remains stable in your blood and easily penetrates into your cells, acting as a brake when CRISPR is too strong.
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