NEW YORK – Mammoth Biosciences has recently forged a novel SNP-detecting enzyme, dubbed CasDx1, to support multiplex CRISPR-based infectious disease variant testing.
In collaboration with a team at the University of California, San Francisco, the firm has demonstrated the new CRISPR-associated systems (Cas) enzyme can detect and distinguish multiple SNPs within a single assay. Mammoth has incorporated CasDx1 into an assay for SARS-CoV-2 variants on its DETECTR system, which the firm hopes will offer labs a faster, cheaper alternative to sequencing-based viral surveillance.
In the Swiss Army knife metaphor of CRISPR technology, Cas enzymes might be likened to the blades or gadgets. Different Cas enzymes are useful for different applications — from gene editing to infectious disease diagnosis — in part due to their functionalities and in part because of their preferred substrates and optimal conditions.
The CRISPR-Cas system is one way bacteria can acquire immunity against invading viruses, and therefore the Earth's microbiome is a great source of new Cas technologies. Tweaking known enzymes to see what happens is another way to create new Swiss Army knife gadgets.
Janice Chen, cofounder and chief technology officer at Mammoth, said in an interview that her team currently combines metagenomic environmental discovery and enzyme modification with protein engineering as part of its internal research and development for both the gene editing and diagnostics sides of the business.
CasDx1 was discovered using a metagenomic discovery approach, Chen said. It is a variant of Cas12 that, after reverse-transcription loop-mediated isothermal amplification (RT-LAMP), can specifically recognize single point mutations and lead to fluorescent probe cleavage.
To find new Cas enzymes, Mammoth gets inputs from public and private data sources on different global environmental samples, Chen said. These go into a computational and bioinformatics pipeline to pluck out any novel CRISPR enzymes, which are then reconstituted in the lab.
At this point, "we don't know how they work, we don't know what they need, but we take them through our experimental pipeline" to screen and determine the enzymatic characteristics and conditions required for them to be active, Chen said.
From there, the enzymes are cataloged by their capabilities and the team starts brainstorming different applications for them, also incorporating the insights of its product team into unique use cases and business opportunities.
"That is the toolbox we are able to continuously grow and evolve," she said. "It's pretty fun."
Consistent with a vision laid out when the firm emerged from stealth mode in 2018, Mammoth has been developing its CRISPR-based disease detection technologies to be amenable for different use scenarios. To this end, the firm has also licensed Cas technologies, for example securing licenses from UC Berkeley to commercialize Cas14 in 2019 and Casɸ in 2020.
The Berkeley team has also recently described a Cas13-mediated system called DISCoVER — short for diagnostics with coronavirus enzymatic reporting — that employs a single-use gravity-driven microfluidic device to perform extraction-free isothermal amplification, T7 transcription, and cleavage of a quenched fluorophore. Chen said, however, that this work is separate from Mammoth's projects.
The pandemic has also served to accelerate CRISPR-based diagnostics. Some recent entrants include Proof Diagnostics, a group at UC San Diego with a Cas13d-based SARS-CoV-2 test, a team at the Broad Institute with a COVID panel that can also detect flu, and a blood test for tuberculosis using Cas12a, for example.
The momentum that is building for rapid point-of-care CRISPR-Cas diagnostics "is pretty exciting to see," Chen said.
Variant genotyping with CasDx1
CasDx1 provides a "powerful way of detecting mutations with a simple and rapid workflow," Chen said.
The Mammoth team was inspired to apply the enzyme to variant detection due in part to a lack of alternatives to whole-genome sequencing (WGS) for SARS-CoV-2 surveillance, she said.
Variant tracking is important because some variants have not responded to therapeutic drugs. However, "we learned from talking to folks in the field that WGS is extremely time consuming and resource intensive, and the turnaround times are incredibly long," she said.
S-gene dropouts in PCR-based assays have also been used as a quick hack to detect the Alpha variant, for example, but the Omicron variant also shares that signature.
And so, between a complex assay and a crude one, "CRISPR is sort of a sweet spot in the middle," Chen said.
Furthermore, compared to PCR-based variant genotyping tests, which have been developed for surveillance by a number of groups, the CasDx1 method also has the advantages of being a bit faster and less complex, Chen said, without the need for a thermal cycler.
In a study published last month in the Journal of Clinical Microbiology, Mammoth and the UCSF collaborators showed the CasDx1 enzyme was able to detect and distinguish three critical mutations in the SARS-CoV-2 virus — namely, the L452R, E484K/Q/A, and N501Y mutations — that can in turn be used to genotype known strains and differentiate Omicron from other variants of interest.
The study first compared CasDx1 to Cas12 enzymes called LbCas12a and AsCas12a, which are commercialized by New England Biolabs and Danaher subsidiary Integrated DNA Technologies, respectively. The team found CasDx1 had the highest fidelity.
Having established that CasDx1 excelled at SNP detection, the team then compared the technology to WGS in a cohort of 304 COVID-19 patient respiratory samples. The assay showed approximately 97 percent SNP concordance and 99 percent agreement for lineage classification.
While other teams use recombinase polymerase amplification (RPA) or PCR pre-amplification, the Mammoth team has chosen LAMP for the variant assay.
With the CasDx1 genotyping assay, "we were able to develop somewhat of a degenerate LAMP design that allowed us to get good coverage of particular regions of the SARS-CoV-2 genome," Chen said, which in turn enabled increased sensitivity.
"If you stack that with the CRISPR protein, that's where you get the specificity to detect specific mutations," she added.
The Omicron variant washed over the US after the team had already put the work on BioRxiv, Chen said, but they were able to quickly reconfigure the assay to add a mutation to call out that variant, too. As it is currently configured, the CasDx1 COVID variant test can detect the Alpha, Delta, Kappa, and Omicron variants. However, the study noted that the data-analysis pipeline for CRISPR-based SNP calling can readily incorporate additional targets.
Charles Chiu, a microbiologist at UCSF and a co-author on the JCM study, said that the collaboration with Mammoth is "a great example of how synergy between industry and academia can help accelerate the rapid development of novel technologies to the clinic."
Chiu, who is also a member of Mammoth's scientific advisory board, engaged his UCSF team to assist with the LAMP primer design for the CasDx1 assay. They also obtained the clinical samples for validation, and collaborated on validating the assay and analyzing the data.
Chen noted that Chiu's expertise was also particularly valuable in refining the workflow for the test.
From his perspective as an infectious disease expert, Chiu said CRISPR-based tests are especially promising for point-of-care and point-of-need testing.
Without the requirement for expensive lab equipment, these tests can be run in settings outside of the traditional clinical laboratory and can be produced at low cost, Chiu said in an email.
The turnaround times for CRISPR-based infectious disease tests can also be in the range of 15 minutes if no pre-amplification is required, or within one to two hours if pre-amplification is required.
And, "Because they are nucleic acid-based tests, the performance with respect to sensitivity and specificity is also generally superior to antigen-based tests," Chiu added.
The UCSF team has also been collaborating with Mammoth Biosciences on other infectious disease CRISPR-based tests, such as for Lyme diseases and other tick-borne infections.
"In the near future, these tests may also be used to identify RNA gene biomarkers that look at the patient's host response to infection, instead of just the pathogen," Chiu said, or to predict severity of diseases like Lyme or COVID.
Overall, it has been a busy year for Mammoth. The company now has more than 170 employees, Chen said, and continues to push both on the diagnostics and therapeutics fronts.
The firm attained unicorn status in September after netting $195 million in two funding rounds. With work supported by the National Institutes of Health's RADx program, Mammoth also obtained an Emergency Use Authorization from the US Food and Drug Administration in January for a high-throughput CRISPR-based COVID test.
Since then, "we've been continuously evolving that assay to increase the simplicity, and reduce the turnaround time and cost," Chen said.
The firm is also pushing ahead on adapting mutation detection for applications outside of infectious diseases, she said. And, the therapeutics pillar of the company has also closed partnerships with biopharma — with Bayer and Vertex — to advance Mammoth's ultrasmall nucleases for in vivo gene editing.
But the funnel for diagnostics and therapeutics opportunities is driven by enzyme discovery. CasDx1 is just one example, Chen said.
"There are a number of other enzymes in our toolbox that we can't disclose today, but can be leveraged in different diagnostics and therapeutic use cases," she said.
The ultimate goal is delivering on the vision of taking these enzymes and transforming them into useful products, she said.
For the CasDx1-based variant detection test, users will likely be labs that don't have capabilities or resources to do WGS on all samples.
The paper focused on validating the workflow for a lab-based assay, Chen said, which could in turn be adapted onto an automated high-throughput system. But, she added, because it doesn't require thermal cycling, the assay could also be adapted into a portable system.
CRISPR systems are also tolerant to sample variation and resistant to inhibitors, which makes them particularly amenable to targeted surveillance of dirty samples, like wastewater for example. These systems may also be useful in settings that require consistent high performance at low cost, for example in public health labs.