In search of a cure for CRISPR: is it possible to reverse changes in the genes?

When the scientists behind the Manhattan project, learned about the destruction of Hiroshima and Nagasaki, the enthusiasm of the enthusiasm was replaced by grim regret. What started as a revolution in physics, mutated into a weapon of mass destruction — which was impossible to defend. From the point of view of biology, CRISPR has the destructive power of this magnitude. And scientists do not want history repeated itself again.

 

CRISPR: nuclear weapons in the world of biology

Just five years after opening CRISPR Agency defense advanced research DARPA has initiated a program “Safe” genes: a collaboration of seven leading experts in the field of editing genes in order to identify a variety of antidotes from the CRISPR and better possibilities of editing genes in space and time.

The point is not to feed public fear of a powerful tool; rather, it is necessary beforehand to see the potential dangers and to find preventive measures or countermeasures. If CRISPR is a biological Pandora’s box, it is already open: in clinics began testing CRISPR on people; in the laboratory, the technology turns into gene drives capable of destroying entire species. The purpose of the Safe program Genes — to find a way, or many ways again to close drawers.

Last week the search for the antidote CRISPR become even more active. The team led by Dr. Amit Choudhary from the Ford Institute at mit, a member of Safe Genes, has developed a platform for screening for the rapid screening of more than 10 000 small chemical substances that reduce the activity of the Cas9 scissors.

The team modified the structure of several promising candidates to further enhance their ability to counteract CRISPR, and created two molecules of the antidote, which prevent the binding of Cas9 to the target DNA and its cutting. During the tests on human cells in Petri dishes molecules were passing through cell membranes and safely destroy the activity of CRISPR within a few minutes.

These drugs are the first candidates — and they can be even more toxic than even the CRISPR improvising Hawking inside the body. Scientists will have to test them on animals to evaluate the efficacy and safety.

But a small drugs that counteract CRISPR, even the earliest, show that titanium CRISPR can be stopped. Platform screening’s turning up the chance to find even more powerful button “cancel”: chemical substances that may one day turn into injections or pills, blocking unwanted activity with the editing of genes, in the medical field or even biological weapons.

“These results provide the basis for accurate chemical control of the activity of CRISPR/Cas9, paving the way for the safe use of such technologies,” says Choudhary.

 

What is already available at the moment?

Choudhary is not the only team Safe is Genes seeking molecule anti-CRISPR.

In 2013, another project member, Dr. Joseph Bondi Genome from the University of California in San Francisco helped found the first drugs anti-CRISPR: large, clumsy proteins that block the Cas9 scissors, not allowing them to learn of the DNA molecule or to contact them. His brilliant idea was to return to natural roots as CRISPR system, a bacterial immune defense against viruses.

In nature, CRISPR allows the bacteria to store “a photograph of the offender” — the viral DNA in its own genome, so when the virus strikes again, scissors Cas can break the virus into pieces. But phages also not mistakes. In the evolutionary race, they also developed genes that create proteins anti-CRISPR, to counteract immune defenses of bacteria.

Turning to the biology of phage anti-CRISPR in 2012, Bondi Denomi discovered several new proteins that greatly inhibit the activity Cas12ato Cas9, which is gaining popularity as a diagnostic tool. Working separately Jennifer Doudna from the University of California at Berkeley, one of the first discoverers of CRISPR and also a member of the project used bioinformatics to track down a handful Cas12a killers that block the activity of editing genes in cultured human cells.

At the time Doudna said that these results “are paving a direct path to the discovery of even more anti-CRISPR of the world of microbes”.

Tiny lights

And yet, proteins anti-CRISPR is not particularly useful switches in the real world.

Protein difficult: they are big and clumsy, so I can’t enter the cells and to bite into the mechanisms of CRISPR. They are sensitive to temperature changes and digestion and do not live particularly long in the body. Many of them become an attractive target for our immune system, which can run annoying — if not dangerous, allergic reactions.

Small molecules typically, such problems have not. They are fast, cheap and their effects are reversible. Don’t want to interfere with CRISPR? Just wait until the molecules are washed away. Effective but very hard to find.

And here will help platform screening Choudhary.

High-performance sift through tens of thousands of chemicals using two tests, looking promising. First, it monitors the segments of DNA being attached to the scissors Cas9 with the help of neon lights. DNA is labelled fluorescent “bulbs” that change polarization when you associate with Cas9, like polarized glasses are changing in the sunlight. This allows the team to quickly track, does the molecule bond with Cas9 DNA.

Second, the system uses automatic microscope, looking for fluorescent signals in the cells, acquired or lost in the process of activity of Cas9. In one study, the researchers used cells that normally glows green, while Cas9 does not cut the gene. Potential drug anti-CRISPR will allow the cell to remain green even in the presence of CRISPR dose.

In the process, scientists have identified a molecule BRD0539, which does not allow Cas9 to contact the target DNA sequence. Drug action was perfectly predictable: the higher the dose, the more he suppressed the activity of CRISPR.

These results already help to reduce the side effects of CRISPR in therapeutic conditions. In the cells dose of the drug rapidly reduced the ability of Cas9 cutting in half, which, in turn, reduces unintentional cut the HBB gene involved in sickle-cell anemia — five times.

It is easy to imagine a future where you can take a pill of BRD0539 — or a more powerful equivalent of the next generation — to temporarily reduce or stop the effect of CRISPR, before he starts racing in your body. The drug, being a small molecule, remains stable in your blood and easily penetrates your cells, acting as a brake when the heart is too strong.

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