CRISPR has become more powerful, thanks to the "switch"

With all its amazing capabilitiesgene editing, mechanically CRISPR resembles a power tool with a broken switch. Just think: the whole CRISPR mechanism is built up in a test tube, and after completion it is always active, turned on, and working. After being introduced to animals or people, CRISPR begins to wander all over the body in search of the target gene, which must be edited or destroyed until it loses its strength and is absorbed by the body.

Weakening of molecular controlA power tool is obviously not the perfect solution: it can overdo it and break non-target genes. And if something goes wrong, there is no immediate way to turn off the mechanism before it causes damage.

At the beginning of this month a team from CaliforniaUniversity of Berkeley tried to tame the beast CRISPR. Using a circular permutation technique, the team reorganized CRISPR into the ProCas9 programmable tool, which is quietly hidden in the cells until external factors awaken it — for example, a viral infection.

This “extra level of security” limits CRISPR editing skills to a subset of cells “for precise cutting,” says study author Dr. David Savage.

Moreover, ProCas9 can potentiallyrespond to logical inputs, such as “and” or “no,” which means that it will be activated only if you follow a specific set of instructions — for example, “this cell is cancerous” or “this cell is infected” will lead to the answer “donate a cell” , which activates CRISPR and instructs it to cut the genes necessary for survival. The study was published in the prestigious journal Cell.

Take control of CRISPR

Let's refresh it a little: CRISPR, as a gene editing tool, is actually a molecular duet. The first part, directing RNA, is tiny bloody hounds that are looking for a target gene.

As soon as the gene is captured, the second component - Cas9- activated for cutting. Unlike guiding RNAs, which consist of genetic letters, Cas9 is a protein. It was with this component that scientists from Berkeley decided to play.

"The broad purpose of our work is to tame and for people to use and to remove unnecessary things that are not related to editing the genome," says Savage.

What can be done with protein? Imagine a very long chain of numbered balls (amino acids) crumpled into a complex three-dimensional structure. Cut the chain and be able to reorganize the beads so that their position on the thread is different from the original one, and put them in a knot or two when you reconnect the protein thread.

That is, in essence, the team did. This trick is called a “circular permutation” and transforms the original protein so that it has a new beginning and end - and it develops into another form. This is a huge molecular-level operation that usually destroys the function of the protein.

The authors say that they were not sure that it would work with something as complex as Cas9.

Surprisingly, Cas9 was almost a test. The team attempted to cut it into multiple points before finding cuts that retained the function of the protein, but in 10% of the cases, the reassembled Cas9 worked almost the same as the original.

There is one smart point here: when reconnecting the protein threads, the team slipped into the molecular “gate” - a small linker - which blocked the cutting ability of Cas9 if the linker itself was not broken.

What broke the chains? A set of enzymes called proteases.

Protease-sensitive linker

Imagine proteases as tiny scissors forsquirrels that swim in the body. Their whole family: good ones help us digest steaks and beans. But there are bad. Cancer cells, for example, pump out their own "evil" proteases, which tear up the surrounding tissues, promoting their growth. Viruses can also secrete viral proteases, which is often necessary for their penetration into multiple cells and tissues. Zika and dengue are among those who use proteases as their weapons, and proteases from plant-infecting viruses help desecrate potatoes and other crops.

But proteases are not cut at will. Rather, each of them is intended only for a small number of amino acid sequences - “zip codes”, which it recognizes and cuts.

This means that the team can puta specific zip code corresponding to a specific protease — for example, from cancer cells — to reorganized Cas9 protein as a linker. Thus, the linker will be cut only in cells that have this specific protease, and therefore will be included only in these cells. Depending on the guide RNA, the team can design an activated CRISPR to cut the genes necessary for survival - and thus kill the cancer cell.

In this sense, the new Cas9 proteins, called ProCas9 (“pro” because of “protease”), turn into tiny spy machines that become deadly after activation.

Altruist Killer

To test the concept, scientists presented Zika-infected cells to their new ProCas9, equipped with RNA guides, trained to find genes that support cell life.

In just a week, the new CRISPR system destroyed infected cells as an “altruistic defense.” Healthy cells remained alive and intact.

This showed that the system remained calm in peacetime, thus limiting the genomic damage to the host, the authors say.

In some experiments, ProCas9 worked just as well, sacrificing cells infected with West Nile virus.

“Although this is a very early confirmation of the concept,it demonstrates the idea that it could be a synthetic immune system, ”says study author Benjamin Oaks. "We created a protein that detects a hidden threat that can be programmed for anything."

The new CRISPR system is unlikely to interfere with ourimmune system. Another advantage of protein rearrangement is that its new ends carry loads better, such as other DNA-modifying enzymes or indicators glowing in the dark.

As if Cas9 got a new superpower, whichallows us to track where the protein is in the cell, or to change the expression of certain genes instead of directly spoiling our genetic material.

The team from Berkeley has already provided for a fewuse cases. ProCas9 will be useful for molecular screening or drug discovery. Or, it may limit DNA cutting to certain cells “after the total delivery of the editing complex to the target tissue or organ”, which will significantly increase the safety profile of the instrument, especially in a clinical setting.

But the most interesting thing about all this is that we don’tare tied to the CRISPR mechanism that nature has endowed us with. These proteins can be carefully optimized and placed in scaffolds that are not found in nature, but have the necessary properties for use in human cells, research or treatment, says Savage.

And what uses do you see for the new tool? Tell us about it in our chat. in Telegram.