ika, meet CRISPR.
CRISPR has been hailed for its potential to fix mutations that cause disease. It has hastened the pace of discovery in research labs around the world. And it is often described as a find-and-replace or find-and-delete technique because scientists can harness it to cut DNA and insert or eliminate genetic “letters” of their choosing.
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But researchers have also been exploring CRISPR’s potential to diagnose diseases. Call it the find-and-alert application.
“It’s an area that’s had a little less attention, and yet offers a lot of interesting opportunities,” said Jennifer Doudna, a CRISPR pioneer at the University of California, Berkeley.
With its bloodhound-like ability to speedily sniff out exact genetic sequences, CRISPR could one day be deployed to identify which bacteria or viral strain is afflicting an individual or circulating in a community, leading to targeted treatments and a quicker understanding of bewildering infectious diseases. And doctors could even eventually use CRISPR as part of their toolkit to reveal what’s fueling a patient’s cancer.
“It’s for the same reason why people use it to do gene editing — it’s extremely specific,” said Guillaume Lambert, an author of the recent Zika research and a visiting scholar at Harvard University’s Wyss Institute. “It’s also universal. Any type of sequence can be programmed into CRISPR and then found.”
Scientists still have lots of developing and honing to do before a CRISPR-based diagnostic test is available for real-world use, and at this point, not many researchers are pursuing that.
CRISPR also may not work for some, or even many, diagnostic purposes. The Food and Drug Administration, for example, explored using CRISPR to monitor the spread of pathogens during food-borne outbreaks, but found it was not as effective as other genetic tools, a spokesperson said.
But scientists say the Zika research, published last month in the journal Cell, appeared to mark the first demonstration of CRISPR being used in a diagnostic. If the quest for CRISPR-based treatments is barreling full speed ahead, then the pursuit of a CRISPR-based diagnostic at least has its engines revving.
Researchers at the Delft University of Technology in the Netherlands, for instance, are surveying how a simple diagnostic test that comes on a strip of paper could incorporate CRISPR to classify bacterial infections in the developing world.
The buzz around gene editing following Doudna’s CRISPR discoveries pulled her away from exploring its diagnostic possibilities for a few years, but she said she is interested in restarting that work. And Caribou Biosciences, a company Doudna cofounded in Berkeley, Calif., is looking at designing CRISPR-based diagnostics.
“You can think of a number of ways [CRISPR’s specificity] could be useful in diagnostics,” Caribou CEO and cofounder Rachel Haurwitz said.
Then again, CRISPR did not initially come to mind for the team working on the Zika diagnostic.
For the first part of the research, the scientists — including James Collins of the Massachusetts Institute of Technology, Keith Pardee of the University of Toronto, and Alexander Green of Arizona State University — adapted a paper-based diagnostic they had developed in 2014 for Ebola.
The test features sensors that, when exposed to RNA from the Zika virus, generate a response that causes yellow dots on the test to turn purple.
Getting the sensors to pick up only the Zika genome was an accomplishment in itself, because existing tests for Zika often react to the related dengue virus as well. The test was not fine-tuned enough, however, to determine which of two Zika strains was in the sample. The African and American strains are so similar that in many places they diverge at only one genetic building block: while a sequence might read as AGTC in one strain, it could be AGGC in another.
CRISPR systems include two key components: pieces of RNA that ferry CRISPR complexes to a specific point in the genome, and proteins that snip the genome. Depending on how they craft this “guide RNA,” researchers can direct the system to a precise letter sequence of their choosing, where the protein will then work its cutting magic.
As the scientists worked to refine their Zika test, Lambert suggested integrating CRISPR to distinguish between two strains. Lambert, who is launching a lab at Cornell University this summer, had been using CRISPR and wanted to find a way to apply it to Zika.
For that experiment, the scientists designed the guide RNA to recognize a sequence present in the American strain but not the African strain. The CRISPR system accurately diagnosed the American strain, cleaving the genome and leaving the yellow dots unchanged.
But when turned on the African strain, the system couldn’t locate the target sequence, so no cut was made. In turn, the sensors were activated and yellow dots turned purple.
The scientists are now hustling to convert their demonstration in the lab into a practical Zika diagnostic that can be used around the world.
The basic test that can separate Zika from dengue should cost just a few dollars and produce a result in a matter of hours. And if the second, CRISPR-based component can distinguish among the strains in the real world, it could help disease detectives track the virus’s spread and learn what different effects different strains cause.
The researchers are also already thinking about what else the CRISPR diagnostic platform could do. They envisage loading CRISPR systems onto what are essentially special pieces of paper and creating rapid tests that can be used in community health settings in the United States, as well as the globe’s most remote reaches.
“Our ultimate goal is to have this as an at-home, point-of-care diagnostic,” one that a primary care provider or even someone without medical training could run, said Collins, the MIT bioengineer. “The challenge I gave the team was, could you make this simple enough that I could use it?”
Collins and the other researchers say CRISPR-based tests could theoretically be used to track different strains of HIV in the developing world, and possibly help physicians match patients to the most effective drugs. Doctors could use such tests in their offices to easily figure out what strains of flu are making people sick each year.
“This is Zika, but it could be used for any sequence if you design it carefully,” said Stan Brouns, a molecular microbiologist at the Delft University of Technology, who was not involved with the Zika research.
Cancer is another field where researchers hope CRISPR can play a role in diagnosis, where knowing a tumor’s precise genetic mutations can help determine treatment. The research is still at a preliminary stage, but experts say doctors might one day combine CRISPR with their knowledge of a tumor’s genetics to root out which mutations are driving a patient’s disease and which are just passengers along for the ride.
“The greatest value right now in cancer for CRISPR is that for any given cancer, we have way more candidate genes or candidate mutations than we really understand,” said Elaine Ostrander, a geneticist at the National Human Genome Research Institute who studies prostate cancer.
Sequencing a tumor already helps experts pinpoint key mutations, which can then be attacked with drugs. But what if the sequencing reveals few mutations or comes up with ones that researchers don’t know much about?
That’s where CRISPR could come in. In lab experiments, scientists have used CRISPR to knock out some of these mutations to see how cells respond. Doing this with a patient’s own cells might yield new information about how a mutation works and could be targeted.
In other experiments, researchers have used CRISPR to add mutations from tumors that have stumped them to cells in mice. Perhaps they could then relate what they learn from the animal’s cancer and response to treatments back to the patient from whom the mutation came.
This research is in its infancy and won’t reach large numbers of patients anytime soon, if ever. And learning the secrets of more mutations won’t mean there are treatments available for these patients.
But Jesse Boehm, a cancer biologist and assistant director of the cancer program at the Broad Institute, which conducts genetics research, said scientists working in this area envision a future of such diagnostic strategies.
“A major challenge for the field that CRISPR helps unlock is to separate the so-called driver mutations from the passenger mutations,” Boehm said. “Those pieces of information, together with the genetic analysis of that tumor, would be incredibly important for a patient’s diagnostic report.”