Cas7-11 made to fit into a single viral vector

The CRISPR protein Cas7-11 can precisely edit RNA and has now been re-engineered to fit into a single viral vector, making it more viable for RNA editing in living cells.

In 2021, researchers from the Massachusetts Institute of Technology (MIT; MA, USA) discovered the first CRISPR enzyme that was able to precisely cut strands of RNA; however, this structure was too large to fit into a single viral vector. Now, the same research group along with researchers at the University of Tokyo (Japan) were able to identify which pieces of the enzyme to remove in order to make this CRISPR protein more compact, whilst retaining its RNA targeting abilities.

DNA gene editing with CRISPR-Cas9 has facilitated advances in basic research and gene editing for therapeutic applications. “There are lots of positives about being able to permanently change DNA, especially when it comes to treating an inherited genetic disease,” explains Jonathan Gootenberg (MIT), who co-led this research with Omar Abudayyeh (MIT) and Hiroshi Nishimasu (University of Tokyo). “But for an infection, an injury, or some other temporary disease, being able to temporarily modify a gene through RNA targeting makes more sense.”

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Until now, Cas13 was the only enzyme that could be used to edit RNA; however, once Cas13 recognizes the target, it shreds up any RNA in the cell along with the cell itself. This makes it effective for detecting the presence of RNA pieces in diagnostic tests but is not suitable for applications where precise RNA cutting is required, for example in therapeutics. Unlike Cas13, Cas7-11 can precisely edit RNA strands.

It was already known that Cas7-11 is a combination of five different enzymes, so the researchers used cryo-electron microscopy to study the structure of this enzyme and its effector complex to understand how it folds together into one functioning unit. “The really fascinating thing about Cas7-11, from a fundamental biology perspective, is that it should be all these separate pieces that come together, but instead you have a fusion into one gene,” says Gootenberg. “We really didn’t know what that would look like.”

Cryo-electron microscopy revealed the structure of Cas7-11 whilst binding to both the target RNA strand and the guide RNA so the researchers could identify the structural components of the enzyme essential to its function. “When looking at the structure, it was clear there were some pieces that weren’t needed, which we could actually remove,” explains Abudayyeh. “This makes the enzyme small enough that it fits into a single viral vector for therapeutic applications.” The re-engineered Cas7-11, which the researchers call Cas7-11S, was found to efficiently target RNA when packaged into a viral vector and delivered into mammalian cells.

Additionally, this analysis of the enzyme’s structure is key to understanding how Cas7-11 performs targeted RNA editing and could highlight other ways to modify its function in future research.

The researchers are now planning studies on therapeutic applications of Cas7-11 as well as studying other bacterial proteins with which Cas7-11 interacts.

“Imagine you could have an RNA gene therapy, and when you take it, it modifies your RNA, but when you stop taking it, that modification stops,” says Abudayyeh. “This is really just the beginning of enabling that tool set.”