If the genome-editing powerhouse CRISPR were a dog, it would be the kind you can train to retrieve everything from Frisbees to slippers to a cold beer.
In research reported on Thursday, scientists trained their puppy to be so discriminating it can tell Zika’s genetic material from dengue’s, the DNA in one kind of antibiotic-resistant “superbug” from that in another, and DNA in cancer cells from DNA in healthy cells — even when that DNA is present in quantities equal to a couple of pinches of salt in Lake Superior.
That achievement, described in Science, could pave the way for quick, easy, cheap, and precise diagnostic tests, including in difficult conditions like those in a developing country experiencing a disease outbreak.
“This is a terrific paper and absolutely fine for a first step,” said Dr. Alex McAdam, a medical microbiologist at Boston Children’s Hospital who was not involved in the research. “They’ve developed a promising method of detecting extremely low concentrations of [genetic material], but the key word is ‘promising’: It’s going to be a long walk from hopeful to clinically useful, and there is a lot to do to demonstrate practicality.”
The scientists, led by bioengineer James Collins and CRISPR pioneer Feng Zhangof the Broad Institute of MIT and Harvard, named their system SHERLOCK (for Specific High sensitivity Enzymatic Reporter unlocking). It works in a standard test tube or on glass fiber paper and can run on body heat, offering hope that it could be used in places with no high-tech lab equipment, such as the 2013-2016 Ebola epidemic in West Africa or last year’s Zika outbreak. In contrast, the current workhorse of DNA detection, called PCR, requires sophisticated lab processes, equipment, and heat to make the reaction run.
The best known CRISPR system consists of a “guide” molecule of RNA that darts around a genome until it finds a precise sequence of chemical “letters” — the DNA nucleotides that are abbreviated A, T, C, and G. It also includes a molecular scissors, which snips out the targeted sections of DNA — in a disease-causing gene, for instance — so that the double helix can repair itself with healthy DNA.
SHERLOCK differs in crucial ways. It starts with molecules that find a particular sequence of DNA in a test tube and rapidly make many copies of it. Other molecules turn the “amplified” DNA into its cousin, RNA. Then a CRISPR enzyme that Zhang and colleagues discovered last year, called Cas13a, gets to work: Unlike better-known CRISPR scissors, it doesn’t stop once it has cut the RNA that scientists target. Instead, it also cuts and cuts and cuts more nearby RNA no matter its sequence, in what the researchers call “collateral cleavage.” All that chopping generates a fluorescent signal that can be detected with a $200 device or, sometimes, the naked eye.
SHERLOCK can pick out unique stretches of the genetic material in Zika and dengue viruses, and tell one from the other, in concentrations as low as 2 molecules in a quintillion (that’s a billion billion) — the pinch of salt in Lake Superior. Unlike PCR, it even works after all its components have been freeze-dried, as well as on a simple paper test. That, too, makes it practical for difficult conditions far from labs.
“That increases the portability of the system,” said Collins, a pioneer in paper-based diagnostics, raising hopes that SHERLOCK could be used in the challenging field conditions of an epidemic. The molecules for a test cost $0.61, the authors say.
Coauthor Pardis Sabeti of the Broad called SHERLOCK a “game changer” for identifying infectious diseases. Being able to detect Zika in urine, rather than more invasive and risky blood tests, is “exactly what the world needs,” she said in a statement. (Sabeti did pioneering work identifying strains of Ebola during the West Africa epidemic.)
By constructing their guide RNA just so, the scientists were able to detect and distinguish from one another two notorious antibiotic-resistant strains of the bacterium Klebsiella pneumoniae. That capability could prove crucial during a hospital outbreak of superbugs, when identifying patients’ infections and knowing which antibiotics might work is a matter of life and death.
Even if two targets differ by only a single nucleotide — one has an A and one has a G, for instance — SHERLOCK can tell them apart. That’s the case with African and American strains of Zika, and two strains of dengue.
Another kind of DNA that differs only slightly is DNA that’s released from tumors into the blood. Called cell-free DNA, it is the target of a multimillion-dollar effort by a spinoff of DNA sequencing company Illumina, which hopes to detect cancer exceptionally early. SHERLOCK could detect tumor DNA when there was 1,000 times more molecules of healthy DNA. That’s thought to be good enough for medical use.
The scientists have filed several US patent applications on SHERLOCK, including for uses in detecting viruses, bacteria, and cancer-causing mutations.
“It’s an impressive publication,” said David Duffy, vice president for research at Quanterix, which is also working to develop ways to detect single molecules. “I get sent claims on an almost daily basis saying this is the next best thing, but this checks everything I look for.”