CRISPR ushered in the era of genomic medicine. A line of powerful agents has been developed from the popular CRISPR-Cas9 for the treatment of genetic diseases. However, there is a last mile problem – these tools need to be efficiently delivered to every patient’s cell, and most Cas9s are too large to be included in popular genomic therapy vectors such as adenovirus-associated virus (AAV). .
In a new study, scientists from Cornell explain how this problem is solved by nature: they determine with atomic accuracy how a system derived from the transposon edits DNA under the direction of RNA. Transposons are mobile genetic elements inside bacteria. The transposon line encodes IscB, which is less than half the size of Cas9 but is equally capable of editing DNA. Replacing Cas9 with IscB will finally solve the size problem.
The researchers used cryoelectron microscopy (Cryo-EM) to visualize the IscB-ωRNA molecule from a high-resolution transposon system. They were able to take pictures of the system in various conformational states. They were even able to create thinner versions of IscB by removing irrelevant details from IscB.
“The next generation of sophisticated applications requires the gene editor to be merged with other enzymes and actions, and most Cas9s are already too big for viral delivery. We face congestion at the end of delivery,” said correspondent Aylong Ke, professor molecular sciences. 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 gene therapy like AAV, then we can be sure they can be delivered, and we can focus research solely on the effectiveness of the editing tool itself.”
CRISPR-Cas9 systems use RNA as a guide for DNA sequence recognition. When a match is found, the Cas9 protein cuts the target DNA in the right place; then it is possible to do surgery at the DNA level to correct 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 being achieved by replacing parts of the Cas9 protein with structured RNA (ωRNA) that is fused to the RNA guide. By replacing the protein components of greater Cas9 with RNA, the IscB protein shrinks to the major chemical reaction centers that cleave 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 microbiology. “Exploring these transposons gives us a new starting point for creating more powerful and accessible gene editing tools.”
Transposons – mobile genetic elements – are thought to have been evolutionary precursors to CRISPR systems. They were opened by Nobel Laureate Barbara McClintock ’23, MA ’25, Ph.D. ’27.
“Transposons are specialized genetic hitchhikers who are constantly integrating into and out of our genomes,” Ke said. “The systems inside bacteria, in particular, are constantly being selected – nature has basically diced billions of times and invented really powerful DNA surgical instruments, including CRISPR. And now, by identifying these enzymes in high resolution, we can use their powers.”
As small as IscB was compared to CRISPR Cas9, researchers believe they will be able to reduce it even less. They have already removed 55 amino acids without affecting IscB activity; they hope to make future versions of this genome editor even smaller and therefore even more useful.
Another motivation for the study was a better understanding of the function of the concomitant RNA leader, said one of the first authors, Chuny Hu, a doctoral student at the Department of Molecular Biology and Genetics. “There are still many mysteries – for example, why transposons use a system controlled by RNA? What other roles can this RNA play?”
One problem that remains for researchers is that, although IscB-ωRNA is extremely active in test tubes, it is not as effective at altering DNA in human cells. The next step in their study will be to use a molecular structure to study their potential causes of low activity in human cells. “We have some ideas, in fact a lot of them, that we want to test in the near future,” Schuler said.
The study was funded by grants received by Ke from the National Institutes of Health. Schuler is supported by the Ministry of Defense as part of a scholarship program for graduates in national defense science and technology. The work of Cryo-EM was carried out with the help of the Cornell Center for Materials Research and the Brookhaven National Laboratory.
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