Looking for an antidote to CRISPR: is it possible to reverse the changes in the genes?

When the scientists behind the Manhattan Project,learning about the destruction of Hiroshima and Nagasaki, enthusiastic enthusiasm was replaced by grim regret. What began as a revolution in the field of physics, mutated into weapons of mass destruction - from which it was impossible to defend. Biologically speaking, CRISPR has the destructive power of a similar magnitude. And scientists do not want history to repeat again.

CRISPR: nuclear weapons in the biology world

Just five years after opening CRISPRThe Agency for Advanced Defense Research DARPA initiated the Safe Gene Program: a collaboration of seven leading world experts in the field of gene editing, aimed at finding a variety of antidotes for CRISPR and improving the possibilities of editing genes in space and time.

The point is not to feed the publicfear of a powerful tool; rather, you need to see potential hazards in advance and find preventive measures or countermeasures. If CRISPR is Pandora’s biological box, it is already open: CRISPR tests in humans have begun in clinics; in the laboratory, technology turns into gene drives that can destroy whole species. The goal of Safe Genes is to find a way, or many ways to slam the box again.

Last week, the search for a CRISPR antidote beganeven more active. A team led by Dr. Amit Choudhary of the Wroth Institute at the Massachusetts Institute of Technology, a member of Safe Genes, has developed a “screening” platform for quickly sifting more than 10,000 small chemicals that reduce the activity of Cas9 scissors.

The team refined the structure of severalpromising candidates to further enhance their ability to counteract CRISPR, and has created two antidote molecules that prevent Cas9 from binding to the DNA target and cutting it. During tests on human cells in Petri dishes, molecules passed through cell membranes and reliably destroyed CRISPR activity for several minutes.

These drugs are the very first candidates - and theymay be even more toxic than even CRISPR, persecuted inside the body. Scientists will have to test them on animals to evaluate their effectiveness and safety.

But small drugs that counteractCRISPR, even the very first, proves that titanium CRISPR can be stopped. With the screening platform, there is a chance to find even more powerful “undo” buttons: chemicals that could one day turn into shots or pills that block unwanted gene editing activities in the medical field or even in biological weapons.

“These results lay the foundation for accurate 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?

Choudhury is not the only one from the Safe Genes team who is looking for anti-CRISPR molecules.

In 2013, another member of the project, Dr. JosephBondi-Denomi of the University of California, San Francisco helped find the first anti-CRISPR drugs: large, clumsy proteins that block the Cas9 scissors, not allowing them to know or contact DNA molecules. His brilliant idea was to return to the natural roots of CRISPR as a system of bacterial immune defense against viruses.

At its core, CRISPR allows bacteria to store“The criminal's photo card” - viral DNA - in its own genome, so that when the virus attacks again, Cas scissors can tear the virus into pieces. But phages also do not miss. In the evolutionary race, they also developed genes that create anti-CRISPR proteins to counteract the immune defense of bacteria.

Turning to the biology of phage anti-CRISPR in 2012year, Bondi-Denomi found several new proteins that widely inhibit the activity of Cas12a - an alternative to Cas9, which is gaining popularity as a diagnostic tool. Separately working Jennifer Dudna from the University of California at Berkeley, one of the first CRISPR discoverers and also a member of the project, used bioinformatics to track down a handful of Cas12a killers that block gene editing activity in human cells.

At the time, Dudna said that these results "pave the way for the discovery of even more anti-CRISPR from the microbial world."

Tiny lights

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

With proteins difficult: they are large and clumsy, so they cannot penetrate into the cells and bite into the mechanisms of CRISPR. 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.have They are fast, cheap and their effects are reversible. Do you want to interfere with CRISPR? Just wait for the molecules to wash out. But effective is very difficult to find.

This is where Chowdhury's screening platform will help.

High-performance system sifts dozensthousands of chemicals, using two tests, looking promising. First, she monitors DNA segments, tied to Cas9 scissors with neon lights. DNA is labeled with fluorescent "light bulbs" that change polarization when linked to Cas9, just as polarized glasses change in sunlight. This allows the team to quickly track whether the molecule breaks Cas9's link to DNA.

Secondly, the system uses automaticmicroscopes that search for fluorescent signals in cells, acquired or lost during the activity of Cas9. In one of the papers, 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 moleculeBRD0539, which does not allow Cas9 to bind to the target DNA sequence. The drug's actions were completely predictable: the higher the dose, the more strongly it suppressed the activity of CRISPR.

These results already help reduce side effects.therapeutic effects of CRISPR. In cells, the dose of such a drug quickly reduced the ability of cutting Cas9 in half, which, in turn, reduced the unintentional excision of HBB - the gene involved in sickle-cell anemia - five times.

It is not difficult to imagine a future in which youYou can drink a pill from the BRD0539 — or the next generation’s more powerful equivalent — to temporarily reduce or halt the CRISPR action before it is peddled in your body. Being a small molecule, the drug remains stable in your blood and easily penetrates your cells, acting as a brake when CRISPR is too strong.

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