NEW YORK (GenomeWeb) – Mammoth Biosciences, a biotech startup cofounded last year by CRISPR researcher Jennifer Doudna, has exclusively licensed the newly discovered Cas14 protein from the University of California, Berkeley, in a bid to broaden the range of possible applications for its CRISPR-based disease detection platform.
The discovery of Cas14 was published in Science last November by a team in which Doudna served as senior author and Lucas Harrington — then a researcher in Doudna's lab and now a Mammoth cofounder and the firm's chief discovery officer — served as first author. Although much research remains to be done, so far it seems that this class of enzymes has several advantages other CRISPR-Cas systems. For one thing, the Cas14 nucleases are exceptionally small at only 400 to 700 amino acids in length. Also, Cas14 doesn't seem to need a specific protospacer adjacent motif (PAM) in order to bind or cleave a target DNA sequence.
"Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping," the study's authors wrote. "Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity."
According to Mammoth, the licensing deal with UC Berkeley will allow the company to add Cas14's single-stranded DNA cutting capability to the Cas12-enabled double-stranded DNA targeting capability and Cas13-enabled single-stranded RNA recognition capability of its CRISPR-based platforms.
Last May, Mammoth Cofounder and CEO Trevor Martin laid out the firm's plans to build the platform, initially using Cas12 and Cas13 to look for DNA and RNA in a bid to detect various diseases. The possibilities, from infectious disease detection to cancer biomarker detection, are almost limitless, Martin said at the time, adding that although the initial focus of the platform is healthcare, future possibilities could include applications for genotyping, agriculture, forensics, the oil industry, and more.
He had also noted that the platform would be able to utilize any CRISPR-Cas protein with a purpose, and that Cas proteins could be multiplexed to detect several nucleic acids, or even DNA and RNA, simultaneously.
With the discovery of Cas14, and Mammoth's licensing of it, Martin's vision seems to be coming to fruition. "We're super excited because this has the potential to be one of the most powerful CRISPR systems out there," he said this week. "It's actually one of the most diverse [CRISPR] systems, extremely diverse compared to Cas9 and others, which is super exciting because that diversity means there could be a lot of different functionality. And we're really interested to exploit that and see how we can leverage it for the tools and products we're creating."
For example, Martin noted that Cas14 has the potential to be a useful diagnostic tool because of its secondary cutting activity. After the protein has cleaved its target, it cuts collateral DNA at a rate that's many orders of magnitude higher than other Cas systems. This effect can be used to amplify a diagnostic's readout, making it easier and clearer to interpret, Martin said.
He added that not requiring a PAM could allow researchers to use Cas14 to target previously untargetable regions of the genome. And, of course, its small size makes it far more optimal for packaging and delivery into the body, which makes Cas14 much more attractive for anyone looking to develop CRISPR-based therapeutics.
Mammoth's researchers are also working to fully characterize Cas14 and its potential uses. For example, the fact that it was discovered in archaea might mean that it has a very different immune profile from other Cas systems. So far, Martin said, that's just speculation that must be borne out through additional research.
"We're obviously commercializing diagnostic tests based on these [CRISPR systems], but a long-term part of our vision is enabling CRISPR for many applications, so we don't want these proteins to not fulfill their promise and go unused," he added. "We're the experts in CRISPR biochemistry and we want to do the work that enables others who are experts in other things to use this protein through interesting applications."
The company's experimentation is also extending into trying various protein multiplexing combinations for different applications. Cas12, Cas13, and Cas14 all have several orthologs that have serve different functions and have unique characteristics. Putting them together in different ways could serve a variety of purposes.
"The beauty of the system is it really depends on the application you're going after. You can either have no multiplexing or you can multiplex different DNA targets or different RNA targets. And one thing that's pretty neat to the system is we can multiplex DNA and RNA together," Martin said. "So, it really depends what you're actually going after. Are you going after an RNA virus or a DNA virus? Are you doing genotyping? You can try and do both. That's really determined by the end product."
He noted that Mammoth is currently working with several partners on different applications for the platform, though he declined to name the partners at this point. "What's exciting about what we're building at Mammoth is that we have this giant toolbox, and for any given thing we need to do, we can just pick up the right tools. We're not pigeonholed. You want to detect DNA and RNA — we can do that. You want to multiplex RNA — we can do that, too," he said. "So, I think that's why partners are interested."
Right now, healthcare is the main focus of the platform and Mammoth's partnerships, he added, but the company is still looking to explore other areas, as it initially aimed to do.
The addition of Cas14 to Mammoth's CRISPR platform may also serve another purpose — differentiating it from a similar platform developed in the lab of Doudna rival and Broad Institute researcher Feng Zhang.
The Broad's SHERLOCK (Specific High sensitivity Enzymatic Reporter unlocking) technology was first described in April 2017, when the Zhang lab reported its efforts to build on research Doudna had done in October 2016 on Cas13a. In March 2018, researchers from Zhang's lab published a paper in Science describing the latest refinements to SHERLOCK: It now uses Cas12a, Cas13a, and Csm6 to detect both DNA and RNA, and now has a much easier-to-interpret paper readout that the Broad compared to a pregnancy test.
Neither the Broad nor Mammoth would comment on the similarities between the two platforms at the time. It's worth noting that Mammoth also plans to develop an at-home version of its disease-detection platform and that the pregnancy test is serving as the company's model for affordability and accessibility.
But adding Cas14 to Mammoth's system — a protein family that no other company will ostensibly have — should set the two technologies apart. And finding new ways to look at CRISPR is also one of Mammoth's goals, Martin said.
Cas14 is really great and we're going to be spending a lot of time developing that system on our platform. But also, we're very focused on finding the novel systems within the existing ones ... and thinking about new functions that CRISPR proteins could have," he added. "That's core to what we're doing — diagnostics is a very interesting twist on the classic way of viewing CRISPR, and we're always looking at CRISPR through a different lens. CRISPR is not just a gene editing tool, so if we start thinking about it that way, what are the other interesting systems we could develop on top of it. That's a big focus of the company."