RNA targeting enzyme extends CRISPR toolkit

Researchers at MIT’s McGovern Institute for Brain Research have discovered a bacterial enzyme that they believe could extend scientists’ CRISPR toolkit, making it easier to cut and edit RNA with the kind of precision that until ‘now was only available for DNA editing. The enzyme, called Cas7-11, modifies RNA targets without damaging cells, suggesting that in addition to being a valuable research tool, it provides a fertile platform for therapeutic applications.

“This new enzyme is like the Cas9 of RNA,” says McGovern scholar Omar Abudayyeh, referring to the DNA-cleaving enzyme CRISPR that has revolutionized modern biology by making DNA editing fast, little expensive and accurate. “It creates two precise cuts and doesn’t destroy the cell in the process like other enzymes,” he adds.

So far, only one other family of RNA targeting enzymes, Cas13, has been developed extensively for RNA targeting applications. However, when Cas13 recognizes its target, it destroys all the RNAs in the cell, destroying the cell along the way. Like Cas9, Cas7-11 is part of a programmable system; it can be directed to specific RNA targets using a CRISPR guide. Abudayyeh, McGovern Fellow Jonathan Gootenberg and their colleagues discovered Cas7-11 through extensive exploration of CRISPR systems found in the microbial world. Their findings were recently published in the journal Nature.

Explore natural diversity

Like other CRISPR proteins, Cas7-11 is used by bacteria as a defense mechanism against viruses. After encountering a new virus, bacteria that use the CRISPR system keep a record of the infection in the form of a small extract of the pathogen’s genetic material. If this virus reappears, the CRISPR system is activated, guided by a small piece of RNA to destroy the viral genome and clear the infection.

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

“Some target DNAs, some target RNAs. Some are very good at cleaving the target but have some toxicity, and some don’t. They introduce different types of cuts, they can differ in specificity, and so on,” says Eugene. Koonin, an evolutionary biologist at the National Center for Biotechnology Information.

Abudayyeh, Gootenberg and Koonin scanned genome sequences to learn more about the natural diversity of CRISPR systems and to exploit them for potential tools. The idea, says Abudayyeh, is to build on the work evolution has already done in engineering protein machines.

“We don’t know what we’re going to find,” Abudayyeh said, “but let’s just explore and see what’s out there.”

As the team scoured public databases to examine the components of different bacterial defense systems, a protein from a bacteria isolated from Tokyo Bay caught their attention. Its amino acid sequence indicated that it belonged to a class of CRISPR systems that use large multiprotein machines to find and cleave their targets. But this protein seemed to have everything it needed to do the job on its own. Other known single-protein Cas enzymes, including the Cas9 protein which has been widely adopted for DNA editing, belong to a distinct class of CRISPR systems, but Cas7-11 blurs the boundaries of the CRISPR classification system. , says Koonin.

The enzyme, which the team eventually named Cas7-11, was attractive from an engineering standpoint because unique proteins are easier to deliver to cells and are better tools than their complex counterparts. But its makeup also signaled an unexpected evolutionary history. The team found evidence that during evolution, components of a more complex Cas machine had fused together to form the Cas7-11 protein. Gootenberg equates this to the discovery of a bat when you previously assumed birds are the only animals that fly, thereby acknowledging that there are multiple evolutionary paths to flight. “It totally changes the landscape of how these systems are thought out, both functionally and evolutionarily,” he says.

Precision editing

When Gootenberg and Abudayyeh produced the Cas7-11 protein in their lab and started experimenting with it, they realized that this unusual enzyme offered a powerful way to manipulate and study RNA. When they introduced it into the cells with an RNA guide, it made remarkably precise cuts, cutting off its targets while leaving the other RNAs intact. This meant that they could use Cas7-11 to change specific letters in the RNA code, correcting errors introduced by genetic mutations. They were also able to program Cas7-11 to stabilize or destroy particular RNA molecules inside cells, which allowed them to adjust the levels of the proteins encoded by these RNAs.

Abudayyeh and Gootenberg also found that Cas7-11’s ability to cut RNA could be attenuated by a protein that appeared likely to also be involved in triggering programmed cell death, suggesting a possible link between CRISPR defense and a more extreme response to infection.

The team showed that a gene therapy vector can deliver the complete Cas7-11 editing system to cells and that Cas7-11 does not compromise cell health. They hope that with further developments, the enzyme could one day be used to edit disease-causing sequences from a patient’s RNA so that their cells can produce healthy proteins, or to reduce the level of disease. ‘a protein that causes harm due to a genetic disease. .

“We believe that the unique way Cas7-11 cuts allows for many interesting and diverse applications,” said Gootenberg, noting that no other CRISPR tool cuts RNA with such precision. “This is yet another excellent example of how these fundamental biology-driven explorations can produce new tools for therapy and diagnosis,” he adds. “And we are certainly still only scratching the surface of what exists in natural diversity.”