Discovery provides the starting point for better genre editing tools

CRISPR heralded the era of genomic medicine. A number of powerful tools have been developed from the popular CRISPR-Cas9 to cure genetic diseases. But there is a last-mile problem – these tools need to be delivered efficiently to every patient cell, and most Cas9s are too large to fit into popular genomic therapy vectors, such as the adenovirus (AAV) virus.

In new research, Cornell researchers explain how this problem is solved by nature: they define with atomic precision how a transposon-derived system edits DNA in an RNA-controlled manner. Transposons are mobile genetic elements inside bacteria. A transposon line encodes IscB, which is less than half the size of Cas9, but also capable of editing DNA. Replacing Cas9 with IscB would definitely solve the size issue.

The researchers used cryo-electron microscopy (Cryo-EM) to visualize the IscB-ωRNA molecule from a high-resolution transposon system. They were able to capture snapshots of the system in various conformational states. They were even able to design slimmer IscB variants and remove non-essential parts from IscB.

“Sophisticated next-generation applications require the gene editor to fuse with other enzymes and activities, and most Cas9s are already too large for viral delivery. We face a traffic jam at the end of delivery,” said the corresponding author Ailong Ke, professor in Molecular Biology. Biology and Genetics at the College of Arts and Sciences. “If Cas9s can be wrapped up in viral vectors that have been used for decades in gene therapy, like AAV, then we can be sure they can be delivered, and we can focus the research solely on the effectiveness of the editing tool itself. »

CRISPR-Cas9 systems use RNA as a guide to recognize a DNA sequence. Once a match is found, the Cas9 protein cuts the target DNA in the right place; it is then possible to perform surgery at the DNA level to repair genetic diseases. Cryo-EM data collected by the Cornell team show that the IscB ωRNA system works in a similar way, where its smaller size is obtained by replacing parts of the Cas9 protein with a structured RNA (ωRNA) fused to guide The RNA. By replacing the protein components of the larger Cas9 with RNA, the IscB protein is reduced to the central chemical reaction centers that cut the target DNA.

“It’s about understanding the structure of molecules and how they perform chemical reactions,” said lead author Gabriel Schuler, a PhD student in microbiology. “The study of these transposons gives us a new starting point for generating more powerful and accessible gene editing tools.”

It is believed that transposons – mobile genetic elements – were the evolutionary precursors of CRISPR systems. They were discovered by Nobel Laureate Barbara McClintock ’23, MA ’25, Ph.D. ’27.

“Transposons are specialized genetic hitchhikers that integrate and differ from our genomes all the time,” Ke said. “Especially the systems inside bacteria are constantly being selected – nature has rolled the dice billions of times and developed some really powerful DNA surgical tools, including CRISPR. And now, by defining these enzymes in high resolution, we can harness their powers. »

As small as IscB is compared to CRISPR Cas9, the researchers believe they will be able to reduce it even more. They have already eliminated 55 amino acids without affecting the activity of IscB; they hope to make future versions of this genome editor even smaller and therefore even more useful.

Better understanding of the function of accompanying guide RNA was another motivation behind the study, said co-author Chunyi Hu, a postdoc researcher at the Department of Molecular Biology and Genetics. “There’s still a lot of mystery – like why do transposons use an RNA-driven system? What other roles can this RNA play?”

A remaining challenge for researchers is that while IscB ωRNA is extremely active in test tubes, it is not as effective at modifying DNA in human cells. The next step in their research will be to use molecular structure to explore the possibilities they have identified for the cause of low activity in human cells. “We have a few ideas, many of them actually, that we look forward to testing in the near future,” Schuler said.

The research was funded by grants Ke received from the National Institutes of Health. Schuler is supported by the Department of Defense through the National Defense Science and Engineering Graduate Scholarship Program. Cryo-EM work was assisted by the Cornell Center for Materials Research and the Brookhaven National Laboratory.

Source of the story:

Materials supplied by Cornell University. Originally written by Linda B. Glaser, courtesy of the Cornell Chronicle. Note: The content can be edited for style and length.

Leave a Comment