CRISPR-on-chip can be a tool for the diagnosis of cancer
CRISPR, child of modern biology, is growing by leaps and bounds. It grew out of an incomprehensible part of the bacterial protective immune system into a tool for the treatment of genetic diseases, improvement of microorganisms to improve food production and pest control. Since then, scientists have adopted this tool to edit genes and began to use it on mammalian cells, CRISPR was barricaded for cell membranes. gene editing technology works its magic by cutting pieces of faulty DNA and inserting a healthy substitute. All these actions of cut and paste segments took place in living cells. Still.
Last week, the CRISPR Journal magazine study was published, finally released the CRISPR from his prison cell. Replacing a component of the "scissors" CRISPR alternative version, the scientists from the Institute of gene editing in Delaware have developed a new CRISPR system that can be cut free floating DNA in a test tube.
To assist in the reaction tube is filled with cell extracts: a set of enzymes and other biomolecules necessary for CRISPR.
What's the breakthrough?
Making CRISPR work outside of the cage may seem strange academic exercise, but scientists had in mind two specific goals when designing a system.
First, this tool allows scientists to simultaneously make multiple genetic insertions, while previous versions were limited to the editing of DNA within a gene. This is great news for personalized medicine, particularly cancer diagnosis; Many cancers have mutations in several places that respond to different treatments in different ways. Before treatment, doctors often send a biopsy sample of the patient tumor DNA sequencing. This important step will help identify many genetic mutations that lead to the growth or spread of particular tumor.
With the new tool, scientists can accurately simulate these mutations into synthetic DNA fragments in vitro, in effect recreating cancer in a safe, controlled environment. This gives scientists access to biological pathways affected by mutations, and can help create a personalized treatment strategy.
What is even more impressive, this kind of diagnosis can be carried out in just one day. "This is particularly important for the diagnosis of cancer-related when the bill goes to minutes or hours," said study author Dr. Eric Kmiets.
Kmiets not the first to see in the CRISPR diagnostic tool. The fact that in addition to gene therapy CRISPR can powerfully to show themselves in the diagnosis, it was clear for a long time. Earlier, we wrote about the fact that two groups of scientists have presented tests DETECTR and SHERLOCK, which effectively hunt Zika virus, dengue or dangerous HPV strains that cause cervical cancer.
Kmiets claims that his invention requires "substantially" less time to confirm the cancer out of the body, mainly because of the ability to make several revisions simultaneously.
Realizing the potential and profitability of the CRISPR as a diagnostic tool, Kmiets and his colleagues are already looking for a commercial partner to develop the technology, "CRISPR-on-a-chip" for the diagnosis of cancer. If you put aside the immediate application research team also hopes to expand the therapeutic potential of CRISPR to a much broader set of human diseases. Existing tools CRISPR ideally suited for the treatment of diseases caused by mutations in a single gene, such as sickle cell disease or Huntington's disease. But since the work Kmietsa aims at several genes at the same time, it could potentially lead to the treatment of diseases with more complex genetic origin - multiple mutations in a variety of genes - if these mutations are well characterized.
through the lens
Insulation CRISPR in vitro has another advantage: it allows scientists to clearly understand what happens before, during and after editing. And because the clinical trials CRISPR actively promoted, it is important to understand how to make technology more accurate and efficient.
CRISPR already achieved a lot, but the inconvenient truth is that scientists are not quite sure how the tool works, getting into the cage. As tools interact with other biocomponents in a cage? He cuts off only the target DNA or his scissors can go hawking in certain circumstances?
"When you work with a CRISPR within a cell, you are working in the black box, which can not watch the mechanisms that make these amazing things," says Kmiets. "You can see the results, that is, changes in the genes, but how do you come to this - not necessarily, but it is important to ensure the safety of CRISPR for the treatment of patients."
Limiting CRISPR a series of biochemical reactions in the test tube, the researchers offer a way to consider the complex molecular interactions that take place during DNA incision replacement genes, and other processes. Approach - exclusively reductionist. But it allows a cell-free system to work like the Arduino, to experiment with the possibilities of CRISPR and create new biological tools, which are difficult to imagine.
gene editing institute almost immediately faced with the problem of developing a cell-free system.
Problem child turned cas9, Scissor protein that is used in the CRISPR systems. When scientists implicated it with plasmid DNA - type circular DNA that scientists are often used to deliver genes into cells - in vitro, the protein was completely inactive.
It turned out that Cas9 be replaced by Cpf1 (aka Cas12a), another member of the growing library of Cas-proteins. Originally opened in 2015, Cpf1 already proven its use in the creation of transgenic mice and correcting mutations that cause muscular dystrophy. Not so long ago Cpf1 used DETECTR system to fight viruses that cause cancer. This protein brighter future: the company Editas, edits genes, licensed for its further development in 2016.
Exchange load. CRISPR-Cpf1 system came into effect in the test tube. In several experiments, the researchers demonstrated that the cell-free system could repeat most editions that CRISPR introduced inside the cell. Remarkably, the scissors have worked a little bit wrong, as in Cas9. When Cas9 makes the cut, it leaves an ultra-smooth "chopped off the ends" to cut DNA. This hampers the introduction of new pieces of genetic material. Since the ends of a very smooth, tools require accurate alignment block replaces the DNA that it is slid into place. On the other hand, Cpf1 leaves "sticky" ends. These DNA pieces act as shoulders, like a tape supporting the grip replacement DNA. Perhaps this is why Cpf1 works better than cas9, in a test tube, but it is yet to be verified.
Kmietsa system - just one example of how far advanced CRISPR. Since CRISPR continues to grow, its developers promise us a lot that we even can not imagine.