Tools CRISPR mastered three new tricks

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2018-03-05 12:00:11

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CRISPR, gene editing superhero, become a bit more powerful. Last week in Science was published three studies in which leading laboratories from around the world presented the latest additions to this technique, converting editor gene detective viruses or keen historian who retells the history of cell DNA. Like CRISPR, the new technology got funny acronyms: CAMERA, DETECTR, and SHERLOCK. A sudden burst of applications of CRISPR clearly shows that scientists have studied, not all potential applications of this technology.

To turn CRISPR into a real multitool, scientists had to thread the needle: CAMERA, cellular recorder, uses rings of DNA to read the history of the cells – for example, to learn about antibiotics or toxins.

SHERLOCK, by contrast, is added to the "sacrificial" molecules of RNA that are cut in the presence of DNA of the virus or cancer to cause a positive signal. It manifests itself in the form of a blue line on the paper strip, as in the pregnancy test.

"All this shows very creative ways used by people to get to work on the discoveries in the field of CRISPR and create these synthetic pathways," says Dr. Dave savage, a protein engineer at the University of California, Berkeley, not involved in the research.

the

Cellular recorder

The Black boxes of the aircraft are a valuable resource for investigators when the flight goes awry. Similarly, scientists have long hoped to invent the cellular time machine, which documents the events in the life of the cell – dose radiation bursts drugs, bouts of inner turmoil that leads to deterioration of health of the cell.

What could be better than to study the history of the cells in DNA?

Dr David Liu of Harvard University took advantage of the ability of CRISPR to accurately cut DNA and developed a "molecular historian" by the name of the CAMERA (CRISPR-mediated analogue multi-event recording apparatus).

Here's how it works. For starters, scientists change the guide CRISPR molecule – the "guide" RNA in bacterial cells to fire only after trigger pull: suppose the input of an antibiotic or other chemical attack. After activation of guide RNA Cas9 prints, scissors CRISPR, in the target location. Without the guide RNA will not be cut.

In addition to the modified CRISPR system, the team also gave the cells two extra piece of DNA encoded in circular DNA called plasmid. Typically, the cell expresses two plasmids with constant speed. But once activated the guide RNA, it (and Cas9) goes for one of the plasmids and eats her, leaving the other untouched.

Therefore, scientists can easily measure the ratio of the two plasmids. Using this system, researchers were able to learn whether the cell is exposed to tetracycline, a known antibiotic.

This first system worked only with bacterial cells. Liu and his team then developed a second technique of CAMERA that uses a modified Cas9. Instead of having to cut the target gene, these scissors find a specific letter of the DNA and change it to another.

As before, the system is activated only in response to certain signals – compounds, nutrients, light or even signaling molecule cells, such as those associated with cancer. The recorder not only records the presence of the signal; reading the percentage of DNA letters that have been replaced, the team can also determine its duration and strength.

Unlike cellular recorders of the previous generation, the system Liu highly sensitive and requires only a dozen cells to generate a strong signal. SCRIBE similar recorder, developed by Dr. Timothy Lu at MIT in 2014, require "in order" more cells to extract the relatively weak signal.

The CAMERA has other useful features. The tool may, for example, to record several signals simultaneously. Data can also be erased using drugs that return the ratio of plasmids to the initial level.

Lou impressed. A new job, he said, "really great" and "important". Although any medical use will require a long journey, this system can help to identify environmental pollutants or to monitor specific molecular signals that convert stem cells into neurons, muscle or other cell types.

the

Hunters viruses

Two other studies devoted to the transformation of the CRISPR into sensitive diagnostic tool.

DETECTR takes advantage of less well-known brother Cas9 – Cas12.

As Cas9, Cas12 follow the guide RNA in the target and cuts it. But the work of the scissors not stopped. Group under the leadership of Jennifer Duddy, one of the inventors of the CRISPR from the University of California, Berkeley, suddenly discovered that Cas12 remains active after removing the target – he immediately begins to hunt for the single-stranded DNA molecules, trying to destroy them.

Watching this curious activity, the researchers decided to add neon molecular signals guide RNA that it glowed bright green after activation Cas12.

To prove the concept, scientists have invented more guide RNAS that bind to different strains of the human papillomavirus (HPV), some of which cause cervical cancer. System DETECTR were able to isolate two particularly dangerous strain of HPV in a broth of various viral strains.

"This protein acts as a reliable tool for detecting DNA from different sources," says study author Janice Chen. "We want to push the limits of technology, which has potential application in any diagnostic situation, where there is a component of DNA, including cancer and infectious diseases".

The Reverse side? Voracity Cas12 may limit its use for the treatment of genetic diseases in humans. Startup Editas already licensed Cas12 for further development, and the new work of scientists can stop his ambitious plans.

Nonetheless, dumping Cas12 from the accounts it is still too early. While human DNA is unwound into single stranded DNA (and becomes a target Cas12), this enzyme is mainly associated with the genomic DNA. This limits its ability to hunt for potential single-stranded victims, explains Dr. Feng Chen of the Broad Institute, who wrote a work on SHERLOCK.

SHERLOCK, based on a system first developed in 2017 – Specific High Sensitivity Reporter unlocking – Cas13 uses as a cutting tool. At Cas13 also has a mode of violence in which he is immersed after the destruction of the original target.

In the case of SHERLOCK Zhang and his colleagues added "sacrificial" RNA molecules that emit a signal after cleavage. In the presence of viral DNA or RNA for example, Zeke, Ebola virus or dengue fever – Cas13 bites viral target RNA and sacrificial, liberating, however, a positive signal.

SHERLOCK 2.0, which was described in Science last week, 100 times more sensitive than the original, and can detect up to four different targets simultaneously.

It is also very practical for field use: all reagents are in a paper strip which is immersed in the test sample. If you get the strip test came back positive – no expensive tools no longer needed.

As DETECTR, it makes SHERLOCK especially useful during outbreaks. The technology can be easily modified to track other DNA molecules in the blood, for example associated with cancer or aging cells.

Together, the studies support a growing research trend of studying the CRISPR applications beyond gene therapy. Compared with therapy based on CRISPR, which requires many years of rigorous checks on safety and effectiveness, these "alternative" methods you could be in research and diagnostic flow much faster.

Although new tricks CRISPR require testing, experts are optimistic.

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