CRISPR-Cas9 is well known, by people who follow science and some who don’t, as the new DNA editing tool that one day might cure genetic diseases or lead to designer babies. Or both.
Whether those headline-grabbing scenarios come true, or CRISPR-Cas9 just remains a useful tool for all kinds of biological research, one thing is for sure. CRISPR-Cas9 does what it does by cutting DNA.
But in a paper published last week in Cell, a group of researchers at the University of California, San Diego, say they have used CRISPR-Cas9 in a very different way. They have tweaked CRISPR-Cas9 to leave DNA alone and instead latch onto RNA molecules—the messengers of genetic instructions—in a live cell, which allows them to track the RNA as it moves around the cell (pictured above). This could, in turn, lead to insights into the behavior of cells themselves and potentially new ways to study or attack diseases.
The possibility of using CRISPR to monitor and even alter RNA got a shout-out Wednesday from billionaire Paul Allen. To push ahead with the RNA work, University of Berkeley, California biochemist and CRISPR-Cas9 pioneer Jennifer Doudna received one of four individual awards of $1.5 million from a new $100 million program funded by Allen. He established his Paul G. Allen Frontiers Group to encourage “out of the box approaches at the frontiers of knowledge,” he said, speaking at an unveiling today in Washington, DC.
Doudna is a coauthor of the Cell paper, in part because the work from the UCSD lab was based on earlier insights her Berkeley lab published in 2014. The UCSD team is led by Gene Yeo, who runs a multidisciplinary biosciences lab at the school. They have already formed a company—the working title is Locana, the Sanskrit word for “sight” or “vision”—to turn their invention into a lab tool others can use just for cells in a dish for now. “We could already hand it off as a diagnostic for researchers to use, but not yet to use in patients,” Yeo says. Much more work is at hand. For example, there are many flavors of RNA; Yeo and colleagues only tested their tracking system on four of them.
Here’s what they, and the folks at the Doudna lab, are working on—and what’s gotten the attention of Allen’s philanthropic team. Hundreds of thousands, perhaps millions of tiny strands of RNA, or ribonucleic acid, zip around a cell at any given time, often ferrying DNA instructions to ribosomes, the cell’s protein factories. Each type of cell—in the brain, in muscles, in the heart—makes different proteins, so their RNA profiles are different, too. Being able to identify and track those RNA fingerprints inside living cells, so to speak, could help answer biological riddles, says Yeo, whose lab also studies neurodegenerative diseases.
“For example, when we transplant neuronal cells into the spinal cord to repair injury, we don’t know where the cells go,” Yeo says, speaking by phone from his native Singapore, where he also teaches at the National University. “If we could identify a cell by its RNA content, we should be able to track where it goes.”
The same goes for cancer cells, Yeo says—which means tracking their RNA might eventually lead to better ways to intercept or modify those cells. But baby steps first. The UCSD work was, by Yeo’s own admission, a limited test to see if three main things were possible: to introduce the CRISPR-Cas9 tool with an extra widget into live cells without causing damage or modifying the RNA; to bind Cas9 to RNA in the right place without it also cutting DNA; and to track the RNA by attaching a fluorescent protein.
The researchers did not try cutting RNA with the modified CRISPR-Cas9 in the live cells, although some of the Doudna lab work “indicates that it may be possible,” says Dave Nelles, a graduate student in Yeo’s lab whom Yeo credits with much of the heavy lifting for the paper.
That extra widget, called a PAMmer (PAM is short for protospacer adjacent motif), adds a bit more to the process of creating the CRISPR-Cas9 tool, which consists of DNA-snipping scissors (Cas9) and a guide (CRISPR) that takes the scissors to the right cutting spot. With a PAMmer added, CRISPR-Cas9 ignores DNA and instead hones in on RNA.
This isn’t the first method to allow the tracking of RNA inside a live cell. But other methods require altering the gene that produces the RNA—in other words, creating a modified RNA that carries a recognizable “tag” in order to track it. “This [method] has the advantage that there is no need to modify the gene for the RNA,” says Dana Carroll, a gene editing expert at the University of Utah. (Carroll is not associated with the published work, although he has testified about previous work on behalf of Doudna’s side in the ongoing CRISPR patent fight.)
Doudna and one of her collaborators, Mitchell O’Connell, are credited as co-authors of the Yeo paper. Their 2014 Nature paper described the possibility of using CRISPR-Cas9 to target RNA in a dish, already extracted from cells—not within cells.
From Paris, where she was accepting a different award this week, Doudna wrote that the Cell paper was the first to show how to target RNA in live cells with CRISPR-Cas9. “We continue to investigate ways to improve efficiency and ease of use,” she wrote.
Doudna received her award from Allen to pursue not only the RNA work but to look beyond Cas9, which is a bacterial protein, for ways to target and modify RNA and DNA. That said, CRISPR-Cas9 has become popular in labs worldwide because it is so easy to use. Instead of rebuilding the protein (the scissors) every time a new cut is needed, something older and more established gene editing systems called zinc fingers and TALENs require, researchers only need to program a new guide that matches up with the DNA or RNA sequence they want to target.
Derived from a defense system bacteria use to fend off attacks from viruses, CRISPR-Cas9 has been shown to work in all kinds of cells. A big patent fight is currently playing out at the U.S. Patent and Trademark Office centered around who was first to make the engineered system work in eukaryotic cells—those more advanced than bacteria or archaea. The patent dispute pits a group led by Doudna against the Broad Institute of MIT and Harvard, where Feng Zhang and colleagues also did early CRISPR-Cas9 work.
The Broad work is licensed to Cambridge, MA-based Editas Medicine (NASDAQ: EDIT), which earlier this year became the first CRISPR-Cas9 company to go public. Doudna was briefly associated with Editas before cutting ties. With her intellectual property, she cofounded Caribou Biosciences of Berkeley, which in turn has licensed CRISPR-Cas9 technology for use in human therapeutics to Intellia Therapeutics, also of Cambridge.
Doudna was one of four scientists to receive a $1.5 million award from Allen’s new initiative. The others were Ethan Bier of UC San Diego for investigation of how new life forms emerge, Jim Collins of MIT for work on synthetic microbes that Xconomy profiled here, and Bassem Hassan of the French Institut du Cerveau et de la Moelle épinière, for work on how differences in individuals can be explored through the lens of neural development.
Allen’s Frontiers Group is also funding new research teams. The first two will be headquartered at Stanford University in Palo Alto, CA, and at Tufts University in Medford, MA. The Stanford group will explore how cells interact with their environment, and the Tufts group will focus on the structure of complex organs.