For all its stunning ability to edit the genes, mechanical CRISPR resembles a power tool with a broken switch. Just think about it: the whole mechanism of the CRISPR built in a test tube, and after completion he was always active, enabled, working. After the introduction of animals or people, CRISPR begins to wander throughout the body in search of the target gene that needs to edit or to destroy, until you lose power and not metabolized by the body.
The weakening of the control of molecular tools, obviously not the perfect solution: he can be overzealous and break non-targeted genes. And if something goes wrong, there is no immediate way to turn off the engine before it will cause damage.
Earlier this month a team from the University of California at Berkeley tried to tame the beast CRISPR. Using the technique of circular permutation, the team reorganized into a programmable CRISPR tool ProCas9 that quietly hides in the cells, until he was awaken by external factors — for example, viral infection.
This “additional security” restrict editing skills CRISPR a subset of cells “for cutting,” says study author Dr. David savage.
Moreover, ProCas9 can potentially respond to a logical input, such as “and” or “no”, which means that it will be activated only under a specific set of instructions — for example, “this cell is cancer” or “this cell is infected” will lead to the answer “to sacrifice”, which activates CRISPR and give him instructions to cut the genes necessary for survival. The study was published in the prestigious journal Cell.
Take control of CRISPR
Let’s have a little recap: CRISPR as a tool for editing genes that actually represents the molecular Duo. The first part, a guide RNA, it is tiny bloody hounds who are looking for the target gene.
Once the gene is captured, the second component Cas9 — activated to perform the cutting. In contrast to the guide RNA, which consist of letters, Cas9 is a protein. With this component, I decided to play scientists from Berkeley.
“The broad objective of our work is to tame and to use people and to remove unnecessary things that are not related to editing the genome,” says savage.
What can you do with protein? Imagine a very long chain of numbered balls (amino acids), crumpled in a complex three-dimensional structure. Cut the chain and can re-arrange the beads so that their position on thread differ from the original, and put them in a knot or two when re-connecting the protein strands.
It is, in fact, made the team. This trick is called a “circular permutation” and converts the source protein so that it has a new beginning and end — and it folds in a different form. It’s a huge operation at the molecular level, which usually destroys the function of the protein.
The authors say that were not sure that would work with something as complex as Cas9.
Surprisingly, Cas9 was almost a test. The team tried to cut it at numerous points before you find cuts that preserve the function of the protein, but in 10% of cases rebuilt Cas9 worked almost the same as the original.
There is one smart thing: when you reconnect the protein strands, the team slipped into a molecular “gate”, a small linker which blocked the cutting ability of Cas9, if the linker has not been broken.
What broke the chain? A set of enzymes, called proteases.
Proteotoxicity linker
Think of protease as scissors for tiny protein that float in the body. Their whole family: good help us digest our steaks and beans. But there are bad ones. Cancer cells, for example, pumping your own “evil” protease, which break the surrounding tissues, promoting their growth. Viruses can also secrete viral protease that is often required for their penetration in multiple cells and tissues. Zika and dengue are among those using protease as a weapon, and proteases from the infecting plant viruses help to contaminate potatoes and other crops.
Protease but not cut to your liking. Rather, each of them is designed only for a small number of amino acid sequences — the “zip codes” that it recognizes and cuts.
This means that the team can put a certain zip code, corresponding to a specific protease — for example, from cancer cells into a 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 CRISPR activated to cut the genes necessary for survival — and thereby kill the cancer cell.
In this sense, the new Cas9 proteins, called ProCas9 (“pro” because of the “protease”), turn into a tiny spy machines that become deadly after activation.
Killer-altruist
To test the concept, the scientists introduced infected cells Zeke your new ProCas9, equipped with a guide RNA, trained to search for genes that support life cells.
In just a week the new CRISPR system destroyed the infected cells as “altruistic protection.” Healthy cells remained alive and intact.
It showed that the system remained calm in times of peace, thus limiting genomic damage to the owner, said the authors.
In separate experiments, the system ProCas9 worked just as well, sacrificing infected with West Nile virus cells.
“Although this is a very early proof of concept, it demonstrates the idea that this can be a synthetic immune system,” says study author Benjamin oaks. “We have created a protein that detects hidden dangers, which can be programmed for anything.”
The new CRISPR system is unlikely to interfere with our immune system. Another advantage of the restructuring of the protein is that its new ends to better carry loads, such as other DNA modifying enzymes or indicators, glow in the dark.
If Cas9 got a new superpower that allows us to track where in the cell is a protein or change the expression of certain genes, instead of directly spoiling our genetic material.
The team from Berkeley had already provided several use cases. ProCas9 will be useful for molecular screening or drug discovery. Or it may restrict the circumcision of DNA defined cells “after editing the overall delivery of the complex to the target tissue or organ” that will greatly enhance the security profile of the tool, especially in a clinical setting.
But the most interesting thing about this is that we are not tied to the mechanism of CRISPR, which nature has endowed us. These proteins can be carefully optimized and put in frames, not found in nature, but with the necessary properties for use in human cells, research or treatment, says savage.
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