Convenient size RNA editing | MIT News

Last year, researchers at MIT’s McGovern Institute for Brain Research discovered and characterized Cas7-11, the first CRISPR enzyme capable of making precise, guided cuts on strands of RNA without damaging cells in the process. . Now, working with collaborators at the University of Tokyo, the same team has revealed that Cas7-11 can be shrunk to a more compact version, making it an even more viable option for editing RNA inside living cells. The new compact Cas7-11 was described on May 27 in the review Cell as well as a detailed structural analysis of the original enzyme.

“When we looked at the structure, it was clear there were parts that weren’t needed, that we could actually remove,” says Omar Abudayyeh, research scientist and McGovern Fellow, who led the new work with Jonathan Gootenberg, Research Scientist and McGovern Scholar. and his collaborator Hiroshi Nishimasu from the University of Tokyo. “This makes the enzyme small enough to fit into a single viral vector for therapeutic applications.”

The authors, who also include former McGovern Institute postdoc Nathan Zhou and Kazuki Kato of the University of Tokyo, see the novel three-dimensional structure of Cas7-11 as a rich resource for answering questions about basic biology. enzymes and reveal other ways to modify its function in the future.

Target RNA

Over the past decade, CRISPR-Cas9 genome-editing technology has given researchers the ability to edit genes inside human cells – a boon for basic research and the development of therapies to reverse mutations pathogenic genetics. But CRISPR-Cas9 only works to edit DNA, and for some research and clinical purposes, editing RNA is more efficient or useful.

A cell retains its DNA for life and passes an identical copy to daughter cells when it duplicates, so any changes to DNA are relatively permanent. However, RNA is a more transient molecule, transcribed from DNA and degraded soon after.

“There are a lot of positives to being able to permanently alter DNA, especially when it comes to treating an inherited genetic disease,” says Gootenberg. “But for a temporary infection, injury or other illness, it makes more sense to be able to temporarily modify a gene through RNA targeting.”

Until Abdayyeh, Gootenberg and their colleagues discovered and characterized Cas7-11, the only enzyme capable of targeting RNA had a messy side effect; when it recognized a particular gene, the enzyme – Cas13 – started cutting out all the RNA around it. This property makes Cas13 effective for diagnostic testing, where it is used to detect the presence of a piece of RNA, but of little use for therapeutics, where targeted cuts are needed.

The discovery of Cas7-11 opened the doors to a more precise form of RNA editing, analogous to the Cas9 enzyme for DNA. However, the massive Cas7-11 protein was too big to fit in a single viral vector – the empty shell of a virus that researchers typically use to introduce gene-editing machinery into patient cells.

Structural overview

To determine the overall structure of Cas7-11, Abudayyeh, Gootenberg and Nishimasu used cryo-electron microscopy, which shines beams of electrons at frozen protein samples and measures how the beams are transmitted. The researchers knew that Cas7-11 looked like an amalgamation of five separate Cas enzymes fused into a single gene, but didn’t know exactly how these parts folded and fit together.

“The really fascinating thing about Cas7-11, from a basic biology perspective, is that all of these separate pieces should come together, but instead you have a fusion into one gene,” says Gootenberg. . “We really didn’t know what it would be like.”

The structure of Cas7-11, caught in the act of binding both its target tRNA strand and the guide RNA, which directs this binding, revealed how the pieces came together and which parts of the protein were essential for recognizing and cutting RNA. This kind of structural information is critical in determining how to get Cas7-11 to perform targeted tasks inside human cells.

The structure also illuminated a section of the protein that served no apparent functional role. This discovery suggested that researchers could suppress it, rearranging Cas7-11 to make it smaller without taking away its ability to target RNA. Abudayyeh and Gootenberg tested the impact of removing different elements from this section, resulting in a new compact version of the protein, dubbed Cas7-11S. With Cas7-11S in hand, they packaged the system into a single viral vector, delivered it into mammalian cells, and effectively targeted RNA.

The team now plans future studies of other proteins that interact with Cas7-11 in the bacterium from which it originated, and also hopes to continue working on the use of Cas7-11 for therapeutic applications.

“Imagine you can have RNA gene therapy, and when you take it, it changes your RNA, but when you stop taking it, that change stops,” Abudayyeh says. “This is really just the beginning of enabling this toolset.”

This research was funded, in part, by the McGovern Institute Neurotechnology Program, K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience, G. Harold and Leila Y. Mathers Charitable Foundation, MIT John W. Jarve (1978 ) Seed Fund for Scientific Innovation, FastGrants, Basis for Supporting Innovative Drug Discovery and Life Science Research Program, JSPS KAKENHI, Takeda Medical Research Foundation and Inamori Research Institute for Science.

Comments are closed.