- Piyush Jain continues to innovate major improvements in CRISPR, the genetic engineering tool by which genomes can be edited.
- He and his collaborators have utilized cryo-electron microscopy to see individual atoms during the editing process.
- Seeing the underlying structure allows bioengineers to fine-tune DNA-guided CRISPR tools to create safer treatments for complex genetic diseases and design highly accurate viral diagnostics.
Piyush Jain, Ph.D., is a master of metaphors.
When asked to explain his groundbreaking research involving CRISPR — the genetic engineering system by which genomes can be edited — he is apt to mention Xerox copies, landline telephones, owner’s manuals, scissors, dinosaur bones or even cupcakes to make the complex subject matter more understandable.
For example, he used metaphorical locks and keys to describe improvements to CRISPR that could allow the tool to target virtually any sequence in an organism’s DNA. And Jain often compares the DNA inside every cell in the human body to a master instruction guide and RNA to Xerox copies of the guide.
Now there’s a hairpin bridge.
In the latest CRISPR breakthrough, the associate professor in the University of Florida’s Department of Chemical Engineering, in collaboration with David Taylor, Ph.D., at the University of Texas at Austin, is utilizing an ultra-powerful imaging technique called cryo-electron microscopy, or cryo-EM, which allows scientists to see individual atoms during the RNA-editing process. The resulting work was published recently in “Nature Structural and Molecular Biology.”
The microscopic images revealed that the engineered DNA does something extraordinary: It folds into a tiny, physical hairpin bridge-like structure that guides the CRISPR tool to the target RNA. Using another metaphor, Jain likens the structural discovery to creating a blueprint that reveals the exact physical mechanism behind this remarkable, heretofore unseen process.
Having revealed the exact structure, scientists are no longer forced to rely on guesswork to improve RNA editing. The physical map, or blueprint, allows bioengineers to intentionally tweak, scale and fine-tune DNA-guided CRISPR tools to create safer treatments for complex genetic diseases and design highly accurate viral diagnostics.
“Engineering and expanding the toolbox we have access to is important,” Jain said. “With the improved DNA-guided CRISPR system and being able to see the exact underlying structure, we’re able to design processes and drugs that are more specific, more efficient and more cost-effective. That’s the overall goal.”
Jain has already added significant tools to the toolbox. His lab recently created the world’s first DNA-guided CRISPR system and completely redefined how CRISPR technology works. The innovation was highlighted in “Nature Biotechnology.”
Typical CRISPR uses tiny strands of RNA to guide the tool to the correct location for the genetic edit. Like a pair of genetic scissors (another metaphor), it opens both strands of the targeted DNA sequence to introduce the modification. The correct location for the edits is found using a sort of genetic GPS system. However, this work describes a flipped system where DNA-guided CRISPR uses a single strand of engineered DNA to guide the tool for RNA editing.
Jain and Taylor show, for the first time, the exact atomic shape of the DNA-guided CRISPR apparatus using a cryo-EM system during the RNA editing process.
“We built a synthetic DNA guide with a tiny hairpin loop at the end,” Jain said. “This loop tricks the CRISPR protein into thinking it’s holding its usual target, allowing it to lock onto and cut RNA safely and accurately. Using high-resolution cryo-EM imaging, we discovered exactly how this DNA trick works at an atomic level.”
The initial results of Taylor’s structural analysis surprised researchers, who expected the DNA guides to be oriented in a different direction.
Jain credits Taylor’s lab at UT Austin with the innovation of using cryo-EM to discover the atomic structure — the blueprint.
“Our lab did the initial wet lab work. We figured out the sequence, gave them the sequence and purified everything,” Jain recalled. “The structural analysis was all done at UT Austin. They got not just the overall structure, but the refined structure. Which amino acids in Cas protein are involved, where they are placed, how these amino acids are interacting with RNA, how this DNA is folded and interacting in the complex.”
Jain, the Exxon Mobil Gator Alumni Faculty in Chemical Engineering, is passionate about his work. In improving the capabilities of CRISPR and in revealing the underlying physical processes, he believes scientists will be better equipped to locate and control disease-causing cellular anomalies, or to use his metaphor, problems with the Xerox copies of DNA.
Metaphors aside, it’s interesting to note that Jain actually produces RNA in his lab using a device called a nucleic acid synthesizer. And for his research, he literally orders DNA from a website.
“DNA, it’s really just information,” he said. “Scientists have figured out how to make DNA outside the cells. We can manufacture it. You can literally assemble the DNA, one piece at a time, whatever sequence you want. You go to a website, you enter your sequence and the next day you get your DNA. It looks like a tiny speck, like there’s nothing in the tube. But if you have a lot of it, it looks like a flake. It has the sequence you need, and it only costs two dollars. Can you imagine that?”
Student researchers in Jain’s lab played a huge role in the work, including graduate students Rodrigo Fregoso Ocampo from the Taylor Lab and Carlos Orosco from the Jain Lab.
The productive collaboration between Jain and Taylor will continue into 2027 — the two, along with Giulia Palermo from UCLA, are co-organizing the CRISPR2027 Conference, a highly regarded international meeting in the genetic engineering space.