Electric Fields Accelerate CRISPR-based COVID Test

The world desperately needs a portable, reliable COVID-19 test that can deliver immediate results. Now scientists at Stanford University say they hope that a new diagnostic assay will fill that void.

In a paper published last week in the journal PNAS, the scientists describe how they used electric fields and the genetic engineering technique CRISPR to build a microfluidic lab-on-a-chip that can detect the novel coronavirus. The test delivers results in about half an hour-a record for CRISPR-based assays, according to the authors-and uses a lower volume of scarce reagents, compared with other CRISPR-based tests in development.

We're showing that we have all the elements required to achieve a miniaturized and automated device with no moving parts," says Juan Santiago, vice chair of mechanical engineering at Stanford, who led the research. He adds, however, that more work lies ahead before their test could be ready for the public.

Creativity with CRISPR

CRISPR-based COVID-19 tests have garnered considerable interest from researchers and the public since the pandemic began. Such tests harness the power of CRISPR/Cas, a tool that makes the manipulation of DNA and RNA simple. The biotech-tool-turned-pop-sensation last month earned its inventors a Nobel Prize in Chemistry.

CRISPR also makes for a nifty diagnostic, because of its ability to hunt down genetic code. If one is testing a nasal swab for COVID, for example, CRISPR will seek out the virus's genetic code in the sample and light up to let you know that it found it.

It works like this: The CRISPR complex is made up of a genetic sequence, called guide RNA, that is designed to match up with a segment of genetic code in the target virus. The complex is equipped with an enzyme, typically one called Cas12, that acts as a pair of genetic scissors.

When the CRISPR/Cas12 complex is exposed to a sample, it finds its target genetic sequence, and its genetic scissors chop it. This triggers subsequent reactions that create a fluorescent signal, indicating the presence of the virus-a positive test result.

Several iterations of CRISPR-based COVID-19 tests have been proposed. The U.S. Food and Drug Administration (FDA) has granted emergency use authorization to at least two such tests. The first, in May, was developed by Sherlock Biosciences and the other, in August, by Mammoth Biosciences. Emergency Use Authorization gives these companies the ability to temporarily deploy their tests during the public health emergency.

But these tests, and others like it, typically take at least an hour to return results. They also require several manual steps and a considerable volume of reagents. Such reagents have been scarce during the pandemic. There have been a lot of supply chain issues," says Ashwin Ramachandran, a graduate student at Stanford and first author of the paper. One key reagent sold by a single company in the UK tends to have a backlog of three to four months, he says.

His team improved upon previous CRISPR-based test designs by adding electric field gradients. The technique controls and accelerates the assay, reducing time, manual steps, and reagents.

The test from the Stanford group is elegantly advancing the current CRISPR/Cas12 assay procedure using electric field and microfluidics technologies to extract nucleic acids and move and concentrate reactions," says Melanie Ott, director of the Gladstone Institute of Virology, who was not involved in the paper.

An electrical engineering approach

Stanford's assay involves three main steps. First, the human sample is placed on the microfluidic chip. Electric fields are applied across the sample to selectively extract and then concentrate any viral RNA, or genetic code. The molecules respond to the electric fields based on their ionic mobility. Called isotachophoresis, the approach automates what would normally be a manually intensive step, says Santiago.

Next, the extracted genetic code is manually pipetted off the chip and into a standard test tube. The tube contains an enzyme that converts RNA to DNA (a process called reverse transcription), and another enzyme that replicates it many times, creating multiple copies of the DNA sequence (a technique called isothermal amplification).

Finally, the amplified genetic material is pipetted back onto the microfluidic chip where the CRISPR enzymatic assay is performed. In this last step, electric fields are again used to concentrate key components, such as the target DNA molecules, and the CRISPR enzyme, into a tiny space smaller than the width of a human hair. This increases the chances that the CRISPR complex and its genetic scissors will interact with the target genes, speeding up the reaction.

This is the first work that actually shows that you can use electric fields in solution to move these CRISPR reagents around the way you want," says Ramachandran.

Accuracy vs. portability

Out of 32 samples known to be negative for the novel coronavirus, the assay produced no false positives. Out of 32 positive samples, the Stanford's assay detected 30 of them. The other two samples had very low levels of viral genetic material-below the test's threshold.

That makes the test about ten times less sensitive than the gold standard assay, called PCR, or polymerase chain reaction. PCR is widely used in diagnosing COVID-19, but involves sending samples to centralized labs, typically increasing the amount of time it takes to get results by at least a day.

It's basically like a trade-off," says Santiago. We're willing to be a little less sensitive, if the assay can be very convenient, portable and field-deployable."

Santiago's assay represents an elegant step forward, but the technique isn't ready for use on the public. Its main limitation is that the middle step involves physically moving a sample from the chip to a tube and back, using a pipet. This reduces its usefulness in the field.

Santiago says he and his team are working on automating the system so that all three steps can be performed on the chip. His group received funding from Ford Motor Company to develop this new system.

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