Discovery offers starting point for better gene-editing tools

ITHACA, NY – CRISPR ushered in the era of genomic medicine. A range of powerful tools have been developed from the popular CRISPR-Cas9 to cure genetic diseases. However, there is a last-mile problem: these tools must be efficiently delivered to every patient cell, and most Cas9 are too large to fit into popular genomic therapy vectors, such as the virus associated with adenovirus (AAV).

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

The researchers’ paper, “Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9”, published in Science.

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

“Sophisticated next-generation applications require the gene editor to be fused with other enzymes and activities and most Cas9 are already too large for viral delivery. We are facing a delivery bottleneck,” said corresponding author Ailong Ke, a professor of molecular biology and genetics at the College of Arts and Sciences. “If Cas9s can be packaged into viral vectors that have been used for decades in the field of gene therapy, like AAV, then we can be sure they can be delivered and we can focus research exclusively on that. effectiveness of the editing tool itself.”

CRISPR-Cas9 systems use RNA as a guide to recognize a DNA sequence. When a match is found, the Cas9 protein cuts the target DNA at 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, with its smaller size achieved by replacing parts of the Cas9 protein with a structured RNA (ωRNA) that is fused to the guide RNA. By replacing the protein components of the larger Cas9 with RNA, the IscB protein is reduced to the central chemical reaction centers, which cut the target DNA.

“It’s about understanding the structure of molecules and how they carry out chemical reactions,” said first author Gabriel Schuler, a doctoral student in the field of microbiology. “The study of these transposons gives us a new starting point to generate 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, PhD ’27.

“Transposons are specialized genetic hitchhikers, fitting in and out of our genomes all the time,” Ke said. “The systems inside bacteria in particular are constantly being selected – nature has basically 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 further. 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 the function of companion guide RNA was another motivation behind the study, said co-first author Chunyi Hu, a postdoctoral researcher in the Department of Molecular Biology and Genetics. “There is still a lot of mystery, like why do transposons use an RNA-guided 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 altering 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, a lot of them in fact, that we’re looking 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 Brookhaven National Laboratory.?

– This press release originally appeared on the Cornell University website

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