RNA targeting enzyme extends CRISPR toolkit

McGavan Fellow Jonathan Gutenberg (left) and Omar Abdaier are in the lab. Credit: Caitlin Cunningham

Researchers at MIT’s McGovern Institute for Brain Research have extended scientists’ CRISPR toolkits to easily cleave and edit RNA with precision that was previously only available for DNA editing. I found. This enzyme, called Cas7-11, modifies RNA targets without harming cells. This suggests that in addition to being a valuable research tool, it provides a suitable platform for therapeutic applications.

“This new Enzyme Omar Abdaier, a McGavan Fellow, mentioned the DNA-cleaving CRISPR enzyme that revolutionized modern biology by making DNA editing faster, cheaper, and more accurate, saying it was “like Cas9 in RNA.” “It creates two precise cuts and doesn’t destroy them. Like other enzymes, cells in the process,” he added.

To date, only Cas13, another family of RNA targeting enzymes, has been widely developed for RNA targeting applications. However, when Cas13 recognizes the target, it shreds the RNA in the cell and destroys the cell on the way. Like Cas9, Cas7-11 is part of a programmable system. You can use CRISPR guides to target specific RNA targets. Abudayyeh, McGovern Fellow Jonathan Gootenberg, and their colleagues discovered Cas7-11 through an in-depth study of the CRISPR system found in the microbial world.Their findings were recently reported in the journal Nature..

Exploring the diversity of nature

Like other CRISPR proteins, Cas7-11 is used by bacteria as a defense mechanism against viruses.After encountering a new virus, bacteria adopting the CRISPR system keep a record of the infection in the form of small fragments of the pathogen. Genetic material.. When the virus reappears, the CRISPR system is activated and guided by pieces of RNA that disrupt the viral genome and eliminate the infection.

These ancient immune systems are widespread and diverse, with different bacteria deploying different proteins to combat viral invaders.

“Some target DNAs, some target RNAs. Some are very efficient at cutting targets, but some are toxic and others are not. They introduce different types of cuts. And the specificity can be different, “says Eugene Koonin. Evolutionary biologist at the National Center for Biotechnology Information.

Abudayyeh, Gootenberg, and Koonin have scrutinized genomic sequences to learn about the natural diversity of CRISPR systems and mine them for potential tools. According to Abudayyeh, the idea is to leverage the work evolution has already done in engineering. protein Machine.

“I don’t know what to find, but let’s find out what’s there,” says Abdaier.

When the team was looking through public databases to look at the components of various bacterial defense systems, proteins from bacteria isolated from Tokyo Bay caught their attention. Its amino acid sequence has been shown to belong to a class of CRISPR systems that use large multiprotein machines to find and cleave targets. However, this protein seemed to have everything it needed to do its job on its own. Other known single-protein Cas enzymes, including the Cas9 protein, which is widely adopted for DNA editing, belong to another class of the CRISPR system, but Cas7-11 blurs the boundaries of the CRISPR classification system. Koonin says.

The enzyme, which the team eventually named Cas7-11, was attractive from an engineering point of view because a single protein was easily delivered to cells and could create better tools than complex counterparts. However, its composition also showed an unexpected evolutionary history. Throughout evolution, the team found evidence that more complex Cas machine components fused to create the Cas7-11 protein. Gutenberg recognizes that this is the same as discovering bats, which birds previously assumed to be the only animals to fly, thereby providing multiple evolutionary paths for flight. “It completely changes the way these systems are considered functionally and evolutionarily,” he says.

Precision editing

When Gootenberg and Abudayyeh produced the Cas7-11 protein in the lab and began experimenting with it, they realized that this unusual enzyme provided a powerful tool for manipulating and studying RNA. When they introduced it into the cell with an RNA guide, it made a very accurate cut and cut the target undisturbed by other RNA. This meant that Cas7-11 could be used to modify certain characters in the RNA code to correct errors introduced by genetic mutations. It was also possible to program Cas7-11 to stabilize or destroy specific RNA molecules in the cell. This allowed us to regulate the levels of proteins encoded by these RNAs.

Abudayyeh and Gootenberg also found that Cas7-11’s ability to cleave RNA could be compromised by proteins that may also be involved in inducing programmed cell death. This suggests a possible link between CRISPR protection and a more extreme response to infection.

The team has shown that gene therapy vectors can deliver a complete Cas7-11 editing system to cells, and that Cas7-11 does not compromise cell health. With further development, they will one day use enzymes to edit disease-causing sequences from the patient’s RNA, allowing cells to produce healthy proteins or harming due to genetic disease. I want to lower the level of protein I’m exerting. ..

“We believe that Cas7-11’s unique method of cleaving enables many interesting and diverse applications,” said Gootenberg, who said that no other CRISPR tool could cleave RNA so accurately. I am. “This is yet another great example of how these basic biology-driven explorations can create new tools for treatment and diagnosis,” he adds. “And we are certainly just scratching the surface of what is in natural diversity.”

New CRISPR-Cas system cuts viral RNA

For more information:
Programmable RNA targeting with the single-protein CRISPR effector Cas7-11 by Ahsen Özcan et al. Nature (2021). DOI: 10.1038 / s41586-021-03886-5

Quote: RNA targeting enzyme is a CRISPR toolkit obtained from on September 20, 2021 (2021, September). 20 days) will be extended

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RNA targeting enzyme extends CRISPR toolkit

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