o one would be surprised if scientists announced tomorrow that CRISPR had leapt tall test tubes in a single bound, but until that happens, fans of the superhero genome-editing system will have to be content with a trio of almost-as-flashy (but potentially more useful) new tricks, all unveiled on Thursday.
Some of the world’s leading CRISPR labs have, independently, tweaked CRISPR — adding bursts of light here and rings of DNA there — in ways that could make it even more of a research powerhouse and, possibly, a valuable medical sleuth, able to detect Zika, Ebola, and cancer-causing viruses, or a cell’s history of, say, exposure to toxins.
The inventions, which, like CRISPR itself, have been given clever acronyms — DETECTR, CAMERA, and SHERLOCK — show that scientists have yet to exhaust CRISPR’s talents. The technology is beginning to look like a Swiss army knife (we told you that was the best metaphor) rather than a mere Word editor.
In fact, its potential utility — and profitability — as a molecular diagnostic tool and biosensor are enticing enough that the inventors of the three new uses of CRISPR have all filed for patents on them, and a fourth lab scrambled to post its own CRISPR-based biosensor invention on the bioRxiv preprint server at the exact second that the others were reported in the journal Science.
It’s not all puppy dogs and rainbows, however. One of the inventions also suggests that, even as companies and universities gear up to test CRISPR to cure diseases in people, its unplumbed depths still hold unwelcome surprises: one CRISPR system being eyed as a therapeutic might have previously unsuspected and possibly dangerous effects.
“We’re finding more and more creative ways to make use of these tools, catching up with the diverse applications” that nature has found for CRISPR, said biologist David Liu of Harvard University and the Howard Hughes Medical Institute, who led the CAMERA study.
A cellular time machine
Biologists have many ways to detect what’s happening in a cell right now, including by sequencing its DNA and measuring what proteins it’s making. A time machine to reveal what a cell was up to in the past has been more elusive.
“There are a lot of questions in cell biology where you’d like to know a cell’s history,” said Liu, such as what made it turn cancerous or what in the environment it was exposed to.
He and Weixin Tang realized they could use CRISPR’s core talent — its ability to change a cell’s DNA by snipping out a specified region — to record if a cell experienced a certain event. “We set out to turn changes in a cell’s state into permanent changes in its DNA,” he said. The result is CAMERA, or CRISPR-mediated analog multi-event recording apparatus.
For a bacterial cell data recorder, the scientists programmed CRISPR-Cas9 so that its key component, a molecule that acts like a bloodhound to find a particular region of DNA, is produced only if the bacterium senses something, such as a drug or chemical. Once this “guide” molecule is produced, it directs the Cas9 cutting enzyme to snip certain rings of DNA in the cell, called plasmids, but not other, nearly-identical rings. The targeted plasmids are destroyed, while the untargeted ones are left alone.
To figure out whether the cell was exposed to a chemical (Liu’s team tried tetracycline and other antibiotics, among others), the researchers measure the ratio of the two kinds of plasmid.
One day, the system might be the basis for biological sensors in the environment. It could sense environmental pollutants, or even water, in remote locations, said MIT’s Tim Lu, who developed a similar cellular recorder, called SCRIBE, in 2014. CAMERA is “an important advance,” Lu said, since it works with smaller numbers of bacteria.
CAMERA 2, another data recorder that works in human and other mammalian cells as well as bacteria, uses a different CRISPR system, one that doesn’t cut DNA but changes one DNA letter, or base, to another. Again, the scientists reported in Science, they programmed this CRISPR to be produced only in certain conditions, such as in the presence of antibiotics, nutrients, viruses, or light. But this time the presence, strength, and even the duration of those signals could be determined by what fraction of the DNA had undergone the one-letter change, which is easily measured by standard lab techniques.
Any medical applications are a long way off, but one day CAMERA or a similar cellular recorder might be able to tell which genes turn on, and when, during embryonic development, and which molecules make embryonic cells turn into various kinds of cells, from neurons to muscle to skin, said Harris Wang of Columbia University, who invented a similar CRISPR-based cellular recorder last year: “You can make a cell chronicle what happened to it.”
As for the name “CAMERA,” Liu said, “in general, we try to avoid cute acronyms.” (His base editor, is called … base editor.) “But I like photography, and the acronym captures what it does, so ….”
A virus hunter
Cas9 is the most famous CRISPR enzyme, but there are many others. One, called Cas12a (named Cpf1 when Feng Zhang of the Broad Institute and his colleagues discovered it in 2015), usually acts like every other CRISPR enzyme: It snips DNA that its bloodhound molecule takes it to.
“But we found something absolutely surprising,” said CRISPR pioneer Jennifer Doudna of the University of California, Berkeley, and HHMI. Once CRISPR-Cas12a has found, attached to, and cut its target, Cas12a’s appetite is apparently unquenched, she and her colleagues reported. “It remains active and able to cut any single-stranded DNA that comes by,” she said in an interview.
There’s good news and bad news.
The good news is that Cas12a’s penchant for indiscriminate DNA shredding enables it to detect certain molecular targets. The Berkeley scientists combined their CRISPR bloodhound with a molecular neon sign, which literally glows when Cas12a starts shredding DNA. By programming CRISPR to find DNA sequences that are unique to different human papillomaviruses (HPV), which cause cervical cancer, the scientists got “DETECTR” (DNA Endonuclease Targeted CRISPR Trans Reporter) to identify different HPV strains in test tubes — without the specialized equipment needed for current virus-detection kits.
That raises hopes that DETECTR could easily be deployed during viral outbreaks in resource-poor countries. And since it can flag any kind of DNA, including from tumor cells circulating in the blood and from fetal cells in the blood of a pregnant woman, it might reveal the presence of cancer or birth-defect-causing mutations more simply than current methods, which rely on DNA sequencing.
Now the bad news.
Most DNA is double-stranded (hence “double helix”). But sometimes a cell’s DNA untwists into single strands. If CRISPR-Cas12a is introduced into cells to, say, eliminate a disease-causing gene, “it begs the question of what would happen, and whether it would trigger undesired gene edits,” Doudna said. Most genome editing companies, including two she co-founded, Caribou Biosciences and Editas Medicine, are using a different CRISPR enzyme, but Editas licensed Cas12a.
It remains to be seen if Cas12a, which is better for some genome-editing uses than other CRISPR enzymes, should be off the table for medical use. There isn’t much single-stranded DNA in cells, Zhang pointed out in an interview, and the hungry enzyme “should be bound to genomic DNA and [thus] restricted as to [its] ability to roam around the cell to find single-stranded DNA targets.”
A simple, fast test for infections
Zhang invented SHERLOCK (Specific High Sensitivity Reporter unLOCKing) in 2017, to detect specific sequences of DNA and RNA and thereby tell whether a blood sample contained, say, Zika or dengue (which produce similar symptoms) or both. Version 2.0 is three times as sensitive, he and colleagues reported in one of the new papers.
By combining several CRISPR enzymes, “we are able to achieve detection of multiple genetic signatures” in a single test, Zhang said. A paper strip, like in a pregnancy test, is dipped into a sample, and if a line appears, the target molecule was detected — no instruments required. SHERLOCK’s inventors, like DETECTR’s, hope it can be used during disease outbreaks, like the current flu season, to make fast diagnoses.
SHERLOCK is “scientifically the most interesting [of the new CRISPR inventions] and has the most commercial upside, too,” said Rodolphe Barrangou of North Carolina State University, co-discoverer of what CRISPR does in nature (it’s a bacterial immune system) and editor in chief of The CRISPR Journal, which debuted on Thursday.