NEW YORK – Mammoth Biosciences is considering several applications, including therapeutic and diagnostic, for a family of novel CRISPR-Cas proteins that were recently discovered by University of California, Berkeley researchers in the genome of a huge bacteriophage.
Having secured an exclusive licensing deal with UC Berkeley for the research, development, and commercialization of these Casɸ proteins, Mammoth is now trying to characterize the full range of their capabilities in order to determine how they can best be used. The company is especially interested in defining the proteins' possible role in safe and efficient in vivo editing systems.
The Casɸ proteins were discovered in the labs of Mammoth cofounder and CRISPR pioneer Jennifer Doudna at UC Berkeley and Chan Zuckerberg Biohub investigator and UC Professor Jill Banfield. Doudna, Banfield, and their colleagues described the proteins in a study published in Science in July, specifically highlighting their hypercompact sizes and their expanded genetic target recognition capabilities relative to Cas9 and Cas12. The researchers noted at the time that the proteins are highly versatile, making them potentially useful for both diagnostic and therapeutic purposes.
Casɸ was found in the genome of a so-called Biggiephage rather than in a bacterial genome. Although Casɸ isn't the first CRISPR-Cas enzyme to be discovered in a bacteriophage, it is the first that has so far only been found in other bacteriophages rather than also in bacteria or archaea, Berkeley researcher and co-first author on the Science study Basem Al-Shayeb told GenomeWeb in July.
In their paper, the researchers investigated three Casɸ orthologs: Casɸ-1, Casɸ-2, and Casɸ-3. They noted that Casɸ is a minimal functional CRISPR-Cas system, comprising a single protein of about 70 kilodaltons and a CRISPR array. It uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids, the researchers said, and is active in vitro and in human and plant cells.
In other words, the protein can develop its own guide RNA and cut the target DNA within the same site, Al-Shayeb said at the time. That means it doesn't need to outsource gRNA production to other enzymes like Cas9 or Cas14 do, and unlike Cas12, it doesn't need to carry additional domains for gRNA development.
The researchers concluded that the protein's small size in combination with its minimal PAM requirement would be particularly advantageous for both vector-based delivery into cells and a wider range of targetable genomic sequences.
Because the protein originated in an environmental bacteriophage rather than a pathogenic or human gut bacteria, patients are less likely to have a preexisting immune response to it, as they might to Cas9, making Casɸ potentially useful as a therapeutic tool. And its ability to cut single-stranded DNA in trans could also make it good diagnostic tool.
Now that the company has licensed Casɸ, the researchers in Mammoth's protein discovery group are characterizing the enzymes in order to determine how they can best be used, and are also engaged in discovery efforts to see if they can detect additional Casɸ orthologs and similar novel as-yet undiscovered enzymes in other Biggiephage clades.
"One of the strengths of Mammoth is that we have that protein development group that we've used to develop Cas14, and we're very excited for that group to also further develop Casɸ," said Mammoth Cofounder and CEO Trevor Martin. "We're going to explore the entire family. I think it has a lot to offer."
The company exclusively licensed Cas14 from UC Berkeley in March 2019, shortly after it was discovered by Doudna and her team. Similar to Casɸ, Cas14 nucleases are exceptionally small at only 400 to 700 amino acids in length. They also don't seem to need a specific protospacer adjacent motif in order to bind or cleave a target DNA sequence, making them highly versatile.
Importantly, Martin said, Casɸ and Cas14 fit into the same overall vision Mammoth has for genome editing — finding more ways to efficiently and safely perform in vivo editing, rather than being forced to default to ex vivo editing.
"With families like Cas14, we really started to unlock the potential for in vivo editing because of things like the small size, being able to really package it in a variety of very effective delivery systems," he said. "Casɸ is also very aligned with that philosophy of enabling new functionalities for these CRISPR proteins so they have better delivery or better multiplexing, or unlocking the ability to deliver some of these exciting next-generation systems like base editing."
Martin noted that the company is researching both possible therapeutic and diagnostic applications for the Casɸ enzymes, but added that there's still a lot of work to be done before they're ready to be used in the clinic, both on their functionality and their potential for undesirable outcomes like off-target effects.
Additionally, the company is currently evaluating possible partnerships with firms that could help it to develop applications using Casɸ, in therapeutics, diagnostics, agriculture, or for other gene editing uses.
"Similar to Cas14, as we are developing these enzymes internally, we are also very interested in working with partners who have theses around use cases for Casɸ as well, and working with them to drive these into the clinic as quickly as possible," Martin said.
Importantly, Al-Shayeb had highlighted in July that this research showed how much is still unknown about the wider biology of CRISPR.
"Even with all our knowledge on bacteria and viruses so far, we are barely scratching the surface regarding what is out there," he said. "We are able to cultivate below 1 percent of the bacteria we know, and a small fraction of the viruses that infect that 1 percent. There is a lot to learn about the biology around us and what these entities can do."
Indeed, Martin said, Mammoth is following up on that as well. Its protein discovery researchers are digging into other Biggiephage clades to see if they're harboring any other useful Cas systems.
"By leveraging this exploration, it really unlocks the exciting new systems that can have a huge impact," he added. "So, we're definitely interested in further characterizing the class of organisms that these systems were found in, and others as well. We cast a very wide net."