Discovery offers starting point for better gene-editing tools

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

In new research, Cornell researchers provide an explanation for 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 within bacteria. A line of transposon encodes IscB, which is less than half the size of Cas9 but equally capable of DNA editing. Replacing Cas9 with IscB would definitely solve the size problem.

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 construct narrower IscB variants by removing insignificant parts from IscB.

“The next generation of fancy applications requires the gene editor to be fused with other enzymes and activities and most Cas9s are already too large for virus delivery. We are facing a traffic jam at the end of delivery,” said Ailong Ke, professor of molecular technology. biology and genetics in the College of Arts and Sciences. “If Cas9s can be packaged in viral vectors that have been used for decades in the field of gene therapy, such as AAV, then we can be sure that they can be delivered and we can focus research exclusively on the effectiveness of the editing tool itself.”

CRISPR-Cas9 systems use an RNA as a guide to recognize a DNA sequence. When a match is found, the Cas9 protein cuts the target DNA in just the right place; it is then possible to operate at the DNA level to fix 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) fused to guide RNA. By replacing protein components in the larger Cas9 with RNA, the IscB protein shrinks to the central chemical reaction centers that cut the target DNA.

“It’s about understanding the structure of molecules and how they perform the chemical reactions,” says lead author Gabriel Schuler, a doctoral student in microbiology. “Studying these transposons gives us a new starting point for creating more powerful and accessible genre editing tools.”

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

“Transposons are specialized genetic hitchhikers, integrated into and splitting out of ours all the time,” said Ke. “Especially the systems inside bacteria are constantly being selected – nature has basically rolled the dice billions of times and come up with 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 shrink it even less. They have already removed 55 amino acids without affecting IscB activity; they hope to make future versions of this through-editor even smaller and thus even more useful.

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

A challenge that still remains for the researchers is that although IscB-ωRNA is extremely active in test tubes, it was not as effective at altering DNA in human cells. The next step in their research will be to use the molecular structure to explore the possibilities they have identified for the cause of the low activity in human cells. “We have some ideas, many in fact, that we are eager to test 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 Fellowship Program. The Cryo-EM work was assisted by the Cornell Center for Materials Research and the Brookhaven National Laboratory.

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Material provided by Cornell University. Original written by Linda B. Glaser, courtesy of the Cornell Chronicle. Note! The content can be edited for style and length.

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