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COVID-19 Pandemic Accelerated Development of CRISPR as a Diagnostic Platform in 2020


NEW YORK – As the COVID-19 pandemic progressed this year, it became apparent that accurate, fast, and inexpensive molecular diagnostics were as important for combating the spread of the SARS-CoV-2 virus as social distancing measures, mask wearing, and contact tracing.  


PCR-based testing remains the gold standard for infectious disease diagnostics due to its high sensitivity and specificitybut the technique generally requires laboratory equipment and trained personnel, and its relatively long turnaround times and high cost became limitations in the early days of the pandemic 


Some academic groups and diagnostics companies turned to a faster, more portable, and less expensive alternative in isothermal amplification techniques such as real-time loop-mediated amplification (LAMP)but these methods don’t always demonstrate the same high level of accuracy as PCR for diagnostic testing.  


Others, meanwhile, have turned to much younger and more exciting method, CRISPR, with some groups starting from scratch with new ideas and others repurposing CRISPR-based diagnostics they'd been developing for other viruses to detect SARS-CoV-2. And unlike PCR testing, it seems that almost no two CRISPR-based tests are alike: most of the newly published assays have improved on the speed or ease of use of previous tests, some are meant to work in one step, others are meant to work with smartphones, and a few have even been combined with various isothermal amplification methods or microfluidics.   


Importantly, the pandemic has highlighted the importance of CRISPR as a diagnostic tool — despite their differences from each other, the CRISPR-based assays all seek to improve on existing diagnostic methods, whether by providing a measure of adaptability, a potential for multiplexed viral testing at the point of care, or lower manufacturing costs.  


The pressures of the pandemic have also injected more funding into CRISPR diagnostics research, increased the pace of existing diagnostics development work, and boosted collaboration between academic groups and industry. 


Melanie Ott, director of the Gladstone Institute of Virology, co-led a team that developed CRISPR-based assay for SARS-CoV-2 that can be read with a smartphone, can quantitatively measure patients' viral loads in addition to testing for infection, and is meant to function as a one-step process without the need for separate RNA extraction or amplification.  


The researchers' work, which was published in Cell earlier this month, was funded in part by a grant from the National Institutes of Health's Rapid Acceleration of Diagnostics (RADx) program. The NIH is funding a range of lab-based and point-of-care tests for the detection of SARS-CoV-2 through RADx grants. 


"I think there's really an unprecedented support for this type of research that was not available before, and I think that has definitely sped up the development [of CRISPR-based diagnostics]," she said"also think that many things that we have developed [in academia] have now been catapulted forward." 


And the benefits will also be of value to future work, she added. The assay that Ott and her colleagues have developed was originally geared toward detecting HIV. Because they've now developed an actual device on which to detect SARS-CoV-2, she believes that will help her group make "quantum leaps forward" when they go back to redevelop the test for HIV. 


And due to the programmable nature of CRISPR, the assay is easily adaptable if another SARS virus comes along. "You just have to change the guides, if it's an RNA virus," Ott said"That's the important part — you can followhopefully, very rapidly with a broader choice of diagnostics and devices that have been now developed." 


Mara Aspinall, a professor of practice at Arizona State University's College of Health Solutions, principal investigator of the college's COVID-19 Diagnostic Commons database, and a self-described “diagnostic evangelist,” concurred with Ott’s sentiments. Not only has the pandemic "unassailably" accelerated the pace of diagnostics testing, she said, but eventually, that momentum will carry through to broader infectious disease testing, starting with the flu.  


Certainly, many of the groups developing CRISPR tests for COVID are already thinking in this direction. In April, researchers at the Broad Institute described a CRISPR-based molecular diagnostics platform that uses microfluidics chips for the detection of viruses in human samples. A single chip can detect a single virus in more than 1,000 samples at a time, or search a smaller number of samples for more than 160 different viruses, including SARS-CoV-2. The researchers originally envisioned their Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN) assay as a multiplexed pathogen detection platform, and only made the adjustment to add SARS-CoV-2 to the panel once the pandemic was declared. 


Researchers at Stanford University, who described a combined CRISPR-microfluidic platform for their SARS-CoV-2 assay in November, also noted the potential of eventually developing a multiplex chip that can test for more than one virus at the same time.  


"Going forward, multiplexing is right," Aspinall said"Right now, there's such a focus on COVID… but I think that ultimately, we have to focus on multiplexing to get timely and efficient answers." 


For Jason Steiner, chief strategy officer at genome engineering company Synthego, while the "dramatic acceleration" in diagnostics development work through the pandemic was partly the result of the urgency of the circumstances, it wasn't the whole cause. Scientific development has generally been accelerating in the past few years, he said, as technologies like big data analytics, machine learning, automation, and genome engineering have converged to result in innovative platforms. 


Indeed, many of the groups that developed assays this year combined the base CRISPR platform with another technology, giving each test some interesting features or advantages. For example, the Broad researchers and the Stanford team both combined CRISPR with microfluidics.  


In April, Broad postdoctoral fellow Cheri Ackerman described the team's microwell chips as being made like a stamp, using rubber poured over a mold. And Broad core member and MIT associate professor Paul Blainey added that the miniaturized approach to diagnostics that the team took was both resource-efficient and easy to implement. 


Similarly, the Stanford researchers combined microfluidics and CRISPR on an injection-molded chip for the detection of SARS-CoV-2. In addition, these researchers used a microfluidic method that was enhanced by an electric field. The chips have no moving parts — instead, the electric fields move liquids around, and move molecules through the liquid. 


Some groups also tried combining CRISPR with isothermal amplification. Startup company Caspr Biotech has developed a COVID-19 assay, the Caspr Lyo-CRISPR SARS-CoV-2 Kit, which combines CRISPR-Cas12 with a lyophilized format of isothermal amplification. The marriage of technologies enables viral detection with minimal external equipment, making it ideal for low-resource environments or use in the field, according to Caspr Cofounder and CEO Franco Goytia. 


And in August, a group of Chinese researchers described their assay, which combined isothermal amplification and CRISPR-Cas13a to detect SARS-CoV-2 with near single-copy sensitivity. The researchers noted that the assay doesn't require a thermocycler to run, cutting the turnaround time to about 40 minutes and making the test another good option for low-resource settings. 


"Perhaps the most interesting thing about [the introduction of CRISPR as a platform technology for molecular diagnostics] is the ability to perform these types of assays without sophisticated laboratory equipment," Steiner said. "The CRISPR diagnostics for COVID have gone through a couple of different iterations… but where that technology is likely going is to be able to eliminate complex laboratory equipment, which brings things like handheld diagnostics really into practical use." 


Indeed, researchers led by Tulane University School of Medicine principal investigator Tony Hu published a paper earlier this month describing a COVID assay similar to the one described by Ott's group — the one-step diagnostic is meant to be read with a smartphone and can quantitatively measure patients' viral loads in addition to testing for infection.  


And the Stanford team is currently developing a handheld device for its electric field-driven microfluidic CRISPR chip that would enable it to be used almost anywhere. 


The diagnostics that are most likely to reach consumers first, however, are those coming from Sherlock Biosciences and Mammoth Biosciences. Both companies have signed partnership deals with other firms — Sherlock Bio with Binx Health and Mammoth with the consumer healthcare arm of pharmaceutical company GlaxoSmithKline — to develop and commercialize rapid CRISPR-based point-of-care or home-use diagnostic tests for COVID-19. 


Both companies currently have Emergency Use Authorization from the US Food and Drug Administration for the versions of their tests for use in high-volume CLIA-certified labs, and both have applied for regulatory clearance for their use in CLIA-waived settings. 


The sheer variety of settings where CRISPR-based tests can be used represents "a fundamental change in the way that molecular diagnostics can be deployed," Steiner said. "Technologies like CRISPR are extraordinarily specific and they are, in effect, programmable for any type of nucleic acid detection. You're going to see RNA detection [and] DNA detection for viruses and infectious diseases. You’ll start to see… the concept of having CRISPR diagnostics be like pregnancy tests, for example, or in a similar format. And that'll be an extraordinary advance in diagnostics." 


For now, both Steiner and Aspinall found it hard to predict where and when the first CRISPR-based SARS-CoV-2 tests will be used. For one thing, Aspinall said, some test developers are still working out the optimal sample type for their tests. Although most studies are conducted on nasopharyngeal swabs, most of the groups said they are looking into additional sample types, such as saliva or oral swabs. Hu and his colleagues specifically developed their test to work on saliva.  


In terms of sample types, Aspinall noted that oral swabs probably represent a marked improvement, if they prove to be feasible. "The anterior nasal swabs are OK, but people don't love messing with their nose,” she saidSo, you could do an oral swab and that would probably be ideal. But I think the data is showing it doesn't have enough sensitivity." 


And while sensitivity is most definitely king, cost and convenience will also play a role in consumers' demands for specific tests, no matter what technology underlies them. 


"I believe that people want home tests because they want to take back control, they want the power in their own hands to decide, 'I want to go out today, I want to visit my grandma and I want to do that now, I want to do tests in my own house,'" Aspinall said. "However, the data needs to be there. I think the American public and certainly researchers have gotten much more sophisticated in analyzing these tests. And so, the competition is clear and the standards are clear, and you have to meet it." 


Whether American consumers will find CRISPR-based tests to be more convenient isunpredictable. Certainly, the researchers developing these assays are doing their best to makepoint-of-care or home-use assays as simplefast (with speeds as fast as 15 minutes), and inexpensive as possible (the Stanford researchers cited potential costs of approximately $4 per diagnostic chip). 


If American industry itself is any indication, CRISPR-based diagnostics could stick around for some time to come. In addition to GSK partnering with Mammoth and Binx working with Sherlock Bio, Ford Motor Company has reached out to the Stanford researchers to talk about manufacturing their CRISPR chips, and Hu and his colleagues have already licensed their fundamental CRISPR assay technology to a company called Nanopin Technologies. 


And while the vaccine rollout would seem to promise the end of the pandemic, Aspinall said CRISPR-based tests still have a very big role to play in tamping down SARS-CoV-2 


"One of the biggest challenges now is predicting the end, and when you talk about the end, does it end in a quick simple way? I think the answer is probably no," she said. "How long will we be aggressively testing? It's so exciting to have the vaccines rolling out. However, all indications are that it will take months. Secondly, we then have to assess how strong the immunity is. But then, most importantly, will the virus mutate such that we're going to have to be vaccinated on a regular basis? Is this going to be the flu or polio, vis-a-vis the vaccines? [So] if these tests don't become relevant for COVID, they have potential for other areas."