NEW YORK – Whether they're just using CRISPR or adding something more to it like isothermal amplification technology, several companies have already recognized the power of the gene editing technology for delivering fast, easy-to-use SARS-CoV-2 diagnostics in a variety of settings.
Now, researchers at Stanford University have developed a test-on-a-chip technology that combines CRISPR with electric field-driven microfluidics that takes about 35 minutes from sample input to produce a result. They've got the Ford Motor Company interested in potentially helping them manufacture the plastic chips, which could be produced through the injection molding process, for about $4 each.
Further, because CRISPR is the underlying technology, the chips can be easily programmed to serve as diagnostics for a variety of pathogens once the COVID-19 pandemic is no longer such an urgent threat through a change in the guide RNA (gRNA) that leads the CRISPR-Cas12 enzyme to its target.
The researchers described their electric field-enhanced microfluidic method in a paper published this month in the Proceedings of the National Academy of Sciences. They used a selective ionic focusing technique known as isotachophoresis (ITP) to achieve an appropriate electric field gradient on the chip and for automated purification of target RNA from raw nasopharyngeal swab samples. ITP has been used in several studies to rapidly extract nucleic acids from a range of biological samples such as urine, blood, and cell lysates, and to accelerate DNA and RNA hybridization reactions, the researchers said.
"Our chips have no moving parts, and we use electric fields to move liquids around. We also use electric fields to move molecules through the liquid," explained Juan Santiago, senior author of the PNAS paper and head of the Stanford Microfluidics Laboratory.
"The way electric fields came into the picture is that we realized that CRISPR molecules, and a lot of these biomolecules like DNA and all the reagents that are involved in these reactions, possess a net charge. They're not neutral," added first author Ashwin Ramachandran, a senior PhD student in Santiago's lab. "So, you can use electric fields to move these [molecules] around in these tiny microfluidic channels. That's the underlying basic physics behind all of this."
They then combined this ITP purification with loop-mediated isothermal amplification (LAMP) and the ITP-enhanced CRISPR assay to detect SARS-CoV-2 RNA in nasopharyngeal samples from COVID-19 patients and healthy controls. The electric field gradients in ITP were used to control and effect the rapid enzymatic activity of Cas12, once it had recognized its target nucleic acid. This was achieved using a tailored on-chip ITP process to co-focus the Cas12 gRNA, a reporter single-strand DNA, and target nucleic acids, creating simultaneous mixing, pre-concentration, and acceleration of the enzymatic reactions, the researchers said. This method has the advantages of consuming a significantly smaller volume of reagents than conventional methods for CRISPR reactions, and can be done in an automated fashion.
"We're able to automate complex steps on a chip so that a computer is controlling the electric fields and the electric fields, in turn, are controlling the molecules inside the chip," Santiago said. "We're trying to achieve something that's highly automated, where a human doesn't have to perform too many steps and interact with the fluidic systems or with the assay system in general. … Our particular brand of electric field also has [other] advantages. Because we can move molecules around with electric fields, we're able to purify the sample starting from a complex sample. And we use an electric field to focus together all the reagents of the CRISPR reaction, and we increase their local concentration by more than a thousandfold, and that improves or speeds up the chemical reaction."
The researchers then applied their method to clinical samples, including SARS-CoV-2 positive and negative clinical specimens, in order to demonstrate its utility and evaluate its performance. As the method is currently performed, they introduced the samples to the chip where the on-chip ITP rapidly extracted the total nucleic acids. Next, RT-LAMP isothermal amplification was performed off-chip on the ITP extract using a water bath, targeting the viral N and E genes and human RNase P genes in separate reactions. In the last step of the protocol, the amplified samples were then reintroduced to the chip, and the ITP performed rapid Cas12-based enzymatic reactions for target detection.
Upon Cas12 binding to the target cDNA of the SARS-CoV-2 viral RNA targets, Cas12 promiscuously cleaves DNA, including reporter single-stranded DNA probes labeled with a fluorophore-quencher pair. This results in an unquenching of the fluorophore and an increase in observed fluorescence. The fluorescent readout is used as the signal that the presence of the virus has been detected.
The researchers evaluated the performance of the ITP-CRISPR detection method on 32 patient samples that were positive for SARS-CoV-2 and 32 that were negative. The ITP-CRISPR method correctly detected 30 out of 32 positive samples, and there were no false positives on the 32 negative samples, for a positive and negative predictive value of 93.8 percent and 100 percent, respectively. Among the positive samples, 28 showed a positive signal for both the N and E genes, while two samples showed a positive signal for only one of the genes.
For now, as they wrote in their paper, the researchers are performing the amplification step off the chip in a tube. But they're currently working on a new version of the chip that would incorporate the amplification step, as well as developing a handheld reader that would allow a variety of users to take advantage of the chip technology.
"Currently, we do the amplification off-chip, but we're working towards integrating that as built on a single chip," Ramachandran said. "A user would load the sample and then use electric fields and on-chip electric field control and temperature control to extract nucleic acids, amplify on-chip, and then also use CRISPR to detect [the target]. That's a work in progress."
As for the device, the researchers are envisioning a small box where a user could merely insert the chip, close the lid, wait 30 minutes, and then receive an email with the results. It could be wireless or read and powered by USB.
"You get the nasopharyngeal swab, you deposit it into your chip, the chip goes into the device, you close the lid. As you close the lid, that would engage some electrodes into the chip and also beneath the chip there might be a strip heater or a small heating element, because you have to incubate the amplification at a little higher temperature. And then inside this little box would be a miniature laser and a miniature photomultiplier tube for detection," Santiago explained. "The [microfluidic] channel is smaller than the width of a human hair."
It can be done, he added. Ten years ago, Santiago and his colleagues miniaturized a microfluidics-based, USB-powered detection device similar to what they're proposing to create now. Though that was for the detection of toxins in water and not for biopathogens, he's confident the same type of thing can be done here.
The chip would be the disposable component, Ramachandran also noted, and would contain all the necessary elements for the reaction. All the user would need to do is buy one reader and then keep buying chips.
The user also wouldn't be limited to just one kind of test or assay. The nature of CRISPR technology makes it relatively simple to change the target of the CRISPR enzyme without changing the overall design of the diagnostic — all that needs to change is the gRNA. Once they develop a version of the technology that contains on-chip amplification, the researchers are envisioning a wide variety of uses and settings for their device, whether that's in doctors' offices, hospitals, labs, or in the field. It can be programmed to detect any number of viruses or pathogens, and they're also working on a version that multiplexes the detection of several targets on one chip.
Santiago said the team is currently in talks with Ford — who are no strangers to the efficiency of the injection molding or assembly line manufacturing processes — about funding and mass production.
"It's injection molding and it's a single layer, so it's not three dimensional layers," Santiago said. "So, it could be made extremely cheap, certainly less than $4" per chip in materials costs.
They also have as-yet unpublished work on a multiplex chip that would be able to handle three reactions at once. In this concept, after the user inserts the sample, the purification and amplification would happen as normal, but there would be separate ports with electrodes shuttling the DNA to and from different electrodes for the CRISPR step, with the aim of finding different targets.
"We've built and tested a chip that looks like a trident, with three detection zones, and that's to [detect] three genes in parallel," Santiago said. "With this electric field, it's possible to bifurcate and aliquot multiple reagents. It may be hard to do, say, 32 of them, because then you need many more electrodes and sensors, et cetera. But small numbers, I think we can. We're now already working on parallel detection on small numbers."
The key is the electric fields, Ramachandran emphasized. "They aliquot out these multiple channels automatically so you can use the same electric fields in an automated fashion to bifurcate the amplification products into the parallel channels, where you can do multiple targeted reactions."
While this technology has immediate implications for the COVID pandemic, it has many potential uses in the future. Santiago said he and his colleagues published their protocols before they finished integrating the amplification step onto the chip because they recognized the applicability of the assay for SARS-CoV-2. For the long term, they're trying to work with Ford on large-scale manufacturing, but they're also open to collaborating with Mammoth Biosciences, Sherlock Biosciences, or any or the larger CRISPR companies, to get the ITP-CRISPR system into a larger platform and improve its sensitivity and speed. In turn, the researchers believe they have a lot to offer any potential collaborator.
"If you just look at the CRISPR part, a typical CRISPR assay takes at least 30 minutes or an hour," Santiago said. "We can have the same sensitivity in four minutes and we use 1,000 times less reagents than they use. So, we basically make the cost of the CRISPR reagents negligible compared to the chip, and the chip will just be a couple of dollars."