Look Ma No Coronavirus!

By Mitchell Kriegman   |   May 7, 2020
Virologist Carolina Arias and her husband Molecular Biologist Diego Acosta-Alvear and their two daughters (photo by Liz Fourie)

CREST is a Crispr Hope in the Testing Crisis

With the United States and the world on the verge of reopening from the global shutdown, there has never been a greater need for effective and reliable COVID-19 testing. While the current methods all have their advantages and drawbacks, they are hampered by shortages, expense, and the high-level expertise of technology required to administer these tests.

Meet CREST (Cas13-based, Rugged, Equitable, Scalable Testing), a startling new COVID test based on a CRISPR gene editing technology from an interdisciplinary microbiology team at our University of California Santa Barbara. In the short timeline of one month the CREST team has created a game-changing new test that offers hope for effective scalable inexpensive testing which is immune from the shortages that all the other current tests have been facing.

Behind this breakthrough is a team of scientists who pulled together this innovation while dealing with the challenges of the stay-at-home order, including virologist Carolina Arias, and her molecular biologist husband and fellow team member Diego Acosta-Alvear. Together they have continued to teach their courses at UCSB and conducted the development of this test remotely, breaking nano-barriers that even Ant-Man would shrink from, while dealing with an even greater undertaking – tutoring two rambunctious young preschool daughters – and all while working at home. There’s nothing like having all your lives and roles collide at once.

CREST is now in the early stages of verification, including a local prevalence study with Cottage Hospital Infectious Disease Specialist Dr. Lynn Fitzgibbons. When fully vetted CREST represents a mobile, highly sensitive and efficient breakthrough.

The deadly litany of COVID testing missteps are well known. Having opted out of the World Health Organization protocol, the CDC’s own test was so contaminated that purified water came up positive for the coronavirus. This debacle was followed by over-hyped supplies, chronic insufficient availabilities to hospitals, and shortages of everything from swabs to reagents to processing labs.

No one wants to say it out loud, but the United States is in a testing crisis, short and simple.

Scientists the world over are in a race to develop new modalities of testing – from surveillance iPhone apps, smart thermometers, to community sewage testing. Meanwhile the FDA has been issuing emergency use authorizations on tests without traditional vetting, grasping at anything that can puncture the unreality of reopening the economy without actual infection data to provide the public a sense of security as America develops a sustainable way to live with the coronavirus.

In Santa Barbara, we are fortunate that social distancing seems to have helped. We also have some of the most brilliant minds in the world working right here at Cottage Hospital and at UCSB.

Virologist Carolina Arias moved with her husband Diego and two daughters, three and six, to Santa Barbara in 2017 to teach at UCSB. Shortly after the pandemic was declared, her five colleagues, all professors at UCSB, met on March 14 to discuss forming a team to set up a testing platform in the UCSB Labs. Six days later, as Governor Newsom announced the shelter-in-place order for California, all the labs were subsequently closed, threatening to shut down progress on the new test concept before work had even begun.

Some of the other researchers who participated in the project seen here at the UCSB Labs include Jennifer Rauch, Eric Valois, Sabrina C. Solley, Friederike Braig, Ryan S. Lach, and Naomi J. Baxter, all from the Department of Molecular, Cellular and Developmental Biology at UCSB

  “When we closed all the labs, all of our other research stopped,” Carolina recalls, “we asked for authorization to do this project.” The UCSB administration was convinced to proceed on a minimal basis.

“We were only allowed a few people, a total of six people from all four labs. Taking into consideration that our labs are normally staffed with between ten to twenty people each, we were really working with a skeleton crew.”

The core team included Molecular Biologist Max Wilson who joined the faculty in 2018, neuroscientist and head of the Neurobiology Lab at UCSB Dr. Kenneth S. Kosik, Ms. Arias, and Diego.

“The assay (test) was developed in about a month,” Carolina adds. “Everyone was working day and night on this. We all bring something very different to the table. The teamwork was amazing.”

As Carolina and her husband Diego have sheltered at home, they both continue to teach their courses and take interdepartmental Zoom meetings while home schooling their two high energy three- and six-year-old daughters. Oh, and developing a game changing CRISPR based genome test for the coronavirus on the side.

“Yeah, it’s pretty interesting,” Carolina remarks in a cascade of laughter. “Both of us are working on this COVID-19 project and having the girls here. It’s a lot of different directions that we have to pay attention to at the same time.”

Daughters chime in, make requests, even while she’s being interviewed, but Carolina seems to have endless good humor, patience, and focus.

“It’s madness here as you can imagine,” she says. “I was worried because here I am presenting to my big virology class, and of course, there could not be a better time to be teaching virology. Diego is teaching a grad class, and sometimes we have meetings at the same time. When our daughters come around, we’re like, okay, just go watch some Sesame Street and we’ll be back.”

At the same time, it’s turned out to be a rich, educational experience for her children and they have the priceless opportunity to be close to their parents and their work in the middle of a critical project during an extremely difficult time.

“I was so concerned about homeschooling,” she recalls, “I printed some coloring pages from a biochemistry coloring book for adults and gave it to my three-year-old and I was like, okay, we’re going to sit down and color something. She said, I want to color DNA. So, I asked, you know how DNA looks?”

Her daughter shuffled through the pages of the coloring book and stopped.

“This is DNA,” she said.

Carolina was astonished.

“So, you know, homeschooling methods can vary. My three-year-old knows the structure of DNA. And my older daughter learned about the pairing of DNA from The Magic School Bus. I’m happy.”

The Virus – A Replication Machine

The coronavirus and every virus, in fact, is just basically fat and protein, or put more scientifically, a nucleic acid molecule in a protein coat. It doesn’t “live” except in conjunction with this terrible way it creates disease. The human body is the perfect environment for it. Its entire drive for existence is to replicate. Its reproduction in the human body sets off these things called cytokines, small proteins released by many different cells in the body including those of the immune system and that’s what gets people in trouble.

Sometimes the body’s response to infection can go into overdrive. In some patients, excessive or uncontrolled levels of cytokines are released. These are called Cytokine Storms which then activate more immune cells, resulting in hyperinflammation. Cytokine storms are a common complication not only of COVID-19 and flu but of other respiratory diseases caused by coronaviruses such as SARS and MERS. They are also associated with non-infectious diseases such as multiple sclerosis and pancreatitis. In some people the inflammation causes scarring in the lungs and the patient can’t recover.

Three Stages of Pandemic

We’ve all heard that testing is important for hospitals to deal with incoming patients, but testing is equally, if not more crucial, to economic recovery. Testing doesn’t cure the coronavirus, but it’s the antidote to the shutdown and allows the country to open up safely without going back into another shutdown.

It’s important to keep in mind the three simple stages of dealing with any epidemic. When a disease takes hold, the first stage in addressing it is research. This is common sense. Is it a virus? How common is it? All this entails testing. The second stage is resourcing. Do we have what we need to fight this disease, which in this case, means our hospitals. Do our hospitals have sufficient supplies, equipment, and protection to safely deal with an epidemic? The third stage is sustainability. How will we live long term with the virus until we have a vaccine, or it goes away?

Right now, in the United States, we’re looking at sustainability, the third stage, without having adequately addressed the first stage, testing. So that’s a nightmare for everybody. With the wild claims and misinformation we hear every day, it creates an unreality as we face putting our lives on the line in reopening.

Current Testing Modes Have Significant Issues

“You cannot fight a fire blindfolded,” WHO Director General Tedros Adhanom Ghebreyesus famously said on March 16, 2020, “and we cannot stop this pandemic if we don’t know who is infected.” 

Currently there are two basic categories of tests marketed under different names that are in use. The first is a PCR test, also known as a swab test, which detects if a person is currently infected. The second type of test is a serology test, which is also known as an antibody test. This test is designed to detect through a blood sample if a person has already been exposed to the coronavirus, by detecting if antibodies are present or not.

The P51 fluorescence visualizer with samples

While both types of tests exist in the United States, there are multiple problems that complicate the situation. Firstly, many of these tests are costly and require specialists and expensive lab equipment. Another issue is the high probabilities of false negatives and positives prevalent with the existing tests. One of the very best molecular tests has a 10% probability of false positives and a 12% probability of a false negatives.

The serology tests can give a false positive due to the many types of other coronaviruses in our environment, one of which is the common cold. Another crucial problem is that elements in the tests have been in short supply. One reason is worldwide demand. Another reason is that so much of our healthcare supplies have the little label “Made in China” on them.

“We don’t make these things domestically. The same with all these masks. Everything was produced, of course, somewhere in Asia,” stated Dr. Henning Ansorg, the Santa Barbara County Public Health Officer.

Reagents – The Underbelly of The Testing Shortages

Both types of test require complex and pure “reagents,” and plenty of them. A reagent is a fancy word for the main ingredient of any chemical-based test. Reagents are the chemical that allows the test to be processed. Put simply, without reagents the government test kits are useless.

“All the major countries in the world are wanting the same thing at the same time,” said Doris-Ann Williams, chief executive of the British In Vitro Diagnostics Association, which represents producers and distributors of the lab tests used to detect coronavirus.

Despite the considerable number of viral epidemics these past two decades like SARS in 2003, MERS in 2012, 2015, 2018, Ebola in 2014-16, 2019 and Zika in 2015-16, most countries still don’t stockpile reagents.

Guess who does? China. But by mid-January, China, the world’s leading maker and exporter of reagents, had also run low on supply as the outbreak spread in Hubei Province and beyond.

With the two major testing modalities dependent on reagents, UC Santa Barbara’s team tackled a new way to develop a test for the virus that did not require a reagent.

CREST – A CRISPR Coronovirus Test

Based on CRISPR gene editing technology, UCSB’s CREST uses the RNA cutting Cas13 protein for the virus. For coronavirus detection, the researchers programmed a different RNA protein to recognize specific sites of the virus’s genome.

Using previously available off the shelf lab technology, the RNA is programmed to give off fluorescence when it is cut after locating the Cas13 enzyme. In simple terms, if a fluorescence is detected in test readings, the virus is present. If there is no fluorescence, then there is no virus present. Without the use of reagents, with a CREST test, COVID-19 can be detected with a high degree of certainty. Still to be vetted and put through trials of course.

Extract Amplify Detect

Like every test before it, CREST takes a sample, a swab, from a subject and then amplifies or grows that sample so there is more of the sample to work with. Where CREST differs is in the method of detection which utilizes the Cas13 protein and florescence.

An Eye to Economy and Adaptability

But this isn’t just another test. It’s also a drive to create a process that doesn’t require expensive equipment, doesn’t need highly trained personnel, and can be deployed easily worldwide and in all socio-economical environments. The recent DIY-Bio movement has made equipment affordable, Bluetooth-enabled, and field-ready.

“We’re using equipment that has been used in the jungle or even on a sidewalk,” Carolina offers.

Once finalized and properly trialed the test will potentially be a cheaper and quicker option. It is currently estimated that each test will cost between $5 and $7.

Testing with Dr. Lynn Fitzgibbons at Cottage Hospital

Indicators and thermocycler that helps grow the sample for testing

While developing this test, Arias is also teaming up with top Cottage Hospital infectious disease specialistDr. Lynn Fitzgibbons on a parallel project. The two are setting out to create a small sampling pool of 1,000 subjects from the community, partly to trial the application of their test and partly to test for prevalence in the community. The trial is using the conventional RT-PCR test as well as UCSB’s new CREST. The two hope to gather more concrete information on the actual presence of the virus in our community among both symptomatic and asymptomatic residents.

“Dr. Arias is an excellent virologist, with the energy, experience, and brilliance to lead us through the most important viral public health challenge of these last thirty years,” Dr. Fitzgibbons offers. “She is also a phenomenal role model for so many women in science, representing the ultimate triple threat: a skilled and brilliant scientist, a devoted and compassionate mother, and a lovely, humble and fun person. I’m excited that my own daughter is growing up in a world with role models like Dr. Arias around.”

“It appears that the UCSB team has produced what Dr. Deborah Birx asked for – a testing breakthrough,” said Joseph Incandela, UCSB Vice Chancellor for Research. “They did it while shifting to teaching remotely, taking care of kids at home, and all the other challenges. This is phenomenal, and I hope that we can now navigate the path of FDA approval quickly, for everyone’s sake.”

Well that’s sure to make Dr. Birx’s neck scarf flap flip. The circumstances have presented a unique opportunity to witness the best of science, and of scientists. This could be a game-changer and it was developed right here in Santa Barbara.

Meanwhile who knows how Carolina’s daughters might change the world. Identifying the DNA genome at three years old, having grown up immersed in so much creativity, innovation, knowledge, and affection, what will she do next? Hopefully Carolina Arias’ curious, irrepressible girls will be part of the first Post-COVID Generation who will remember today as a long past chapter that changed the world in significant ways. So many changes we will spend our time adjusting to, they will have already absorbed.

 

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