COVID-19 Testing Gets Crisper with CRISPR-Based Diagnostics

An ideal infectious disease test is accurate, fast, cheap, and can be performed at home. It’s rare for one type of diagnostic test to meet all those criteria but CRISPR technology may pave the way, especially for COVID-19.

On May 6, Sherlock Biosciences was the first company to gain emergency use approval (EUA) for a CRISPR-based kit for detection of coronavirus genetic material. Mammoth Biosciences was second, receiving its EUA for a CRISPR-based protocol and set of reagents on August 31.

As of October 5, EUAs have been granted to 176 companies and institutions for their in vitro molecular diagnosis procedures for SARS-CoV-2. The FDA grants COVID-related EUAs for assays achieving sensitivity of 95% and specificity of 95%, according to Rachel West, Ph.D., a post-doc who now focuses on the pandemic testing as part of her fellowship at Johns Hopkins University’s Center for Health Security.

CRISPR-based assays are said to be as accurate as PCR genetic tests, which are the gold standard for accuracy in COVID-19 diagnostics. In addition, CRISPR tests reportedly also deliver faster sample-to- answer results and rely on simpler equipment. In the words of Janice Chen, Ph.D., CTO at Mammoth, “CRISPR-based diagnostics are accurate, rapid, and require low infrastructure. Plus, they have the potential to be developed into rapid, at-home applications.”

From gene editing to diagnostic applications

CRISPR, which is well known for its gene-editing applications, was pioneered by Feng Zhang and Jennifer Doudna around a decade ago. However co-opting bacterial CRISPR-Cas systems for diagnostics only hit the scene a few years ago when companies such as Sherlock and Mammoth formed. They validated their approaches against viruses like Zika and HPV. Zhang co-founded Sherlock Biosciences and Doudna is a co-founder of Mammoth Biosciences.

When leveraging CRISPR technologies for diagnostic use, researchers need to design primers for an initial isothermal, non-PCR amplification step, and then determine the guide RNA sequence that needs to correspond to appropriate sequences from the ‘foreigner’ being detected, such as coronavirus. Then, a Cas enzyme is modified so that its cutting activity becomes detectable. Mammoth’s innovation was to convert Cas12 to more than a cutter—to make it a signal reporter too so that when it cuts, it also adds a flourescent signal that can be detected by standard lab fluorimeters.

Chen said that Mammoth selected Cas12 because it is readily available from vendors. “In February 2020 when we saw the pandemic coming, we released a white paper on our protocol that includes links to vendors so anyone can buy the components off the shelf and assemble their own CRISPR-based COVID-19 test.”

Mammoth’s EUA is for a pre-packaged kit of the reagents, including their modified Cas12 and guide RNA. It now includes better performing enzymes since the company has improved their design since February. “The EUA is for a lab-based, first iteration of our product. Our primary product launch however will be on a high-throughput version we’re designing with funding from NIH’s RadX program that aims to identify and accelerate innovative COVID diagnostics to market.” In the coming months, Mammoth also hopes to launch a home-based test using lateral flow assays that it is developing with GSK.

Sherlock’s product, as described on their website and in the EUA, uses a modified Cas13 to produce its fluorescent signal once coronavirus genetic material is detected by their guide RNA. Similar to Mammoth, the company says that its EUA version can be quickly adopted by most diagnostic laboratories because it relies on standard lab equipment. Sherlock is now focused on developing an inexpensive, paper-based point-of-care assay which was described in a May 8 preprint.

Point-of-care applications

Another big advantage of CRISPR-based protocols, besides their accuracy being on par with PCR, is the fact they can be performed at one temperature using a type of isothermal genetic amplification called LAMP (loop mediated isothermal amplification). PCR uses thermocyclers, which are hard to adapt for point of care (POC) as trained personnel are required for use.

Current LAMP-based genetic testing provides fast results and has the POC advantage but accuracy is impacted. A well-known example of a nonPCR, LAMP-based POC genetic COVID test is Abbott’s ID Now system, which is used by the White House. CRISPR ,however, does not have the accuracy drawback. As West explained, “CRISPR gives the advantage of precision—you only amplify what you’re interested in. With the other LAMP procedures, there is a possibility that spurious by-products are also amplified."

A few PCR-based diagnostic product companies recently launched POC machines but those products are still based on specialized innovations such as combining mass spectrometry for the readout and are relatively costly. CRISPR-based assays can more easily be adapted to POC. For example, the University of California San Francisco is currently offering Mammoth-based genetic COVID testing. Mammoth and Sherlock as well as others that have published recently are devising POC applications and even at-home applications.

Will CRISPR replace PCR?

The main advantage of PCR-based diagnostics is their accuracy and the fact that powerhouse, high-throughput (HTP) equipment exists because the technology has been around for over 20 years. For example, Roche’s cobas system reportedly has a throughput of approximately 3,000 samples/24 hours.

Now that CRISPR has proven to be as accurate as PCR, the next challenge is to increase CRISPR’s throughput such that it can be used in reference labs providing results on many samples/day. Mammoth expects to launch its HTP product in early 2021, and Chen says it should be able to provide results in one hour versus three to fours hours for PCR HTP machines, so its throughput could be higher than PCR.

Gene Civillico, Ph.D., a program manager at the NIH and part of the team that manages the ‘Shark Tank’-like RadX Technology initiative that grants funding to COVID-19 diagnostic innovators, including Mammoth, believes the diagnostic testing scene is wide open. “There are innovators, for example, who are shrinking PCR testing to toaster oven size so as to combine accuracy with ease and point of care possibilities.” Civillico does admit that CRISPR applications are unique in that they combine speed and ease with accuracy. “With CRISPR you get the same sensitivity as PCR but in less time.”

Chen offers a realistic picture of the challenges ahead for CRISPR: “It’s hard to compete with a two-year technology against the familiarity, improvements, and infrastructure that accompany a 20-year old technology such as PCR. We have a lot of innovating ahead of us; though we are at the forefront. There is much more research coming from academic labs as well as ours. For example, figuring out the best conditions for CRISPR-Cas enzymes to tolerate different conditions such as the increased viscosity of saliva or other at-home-compatible sample preps are in the works.”