This article has been updated to clarify the length of the Cas13d protein as referred to in the study by Konermann et al.
NEW YORK (GenomeWeb) – With $15.6 million in Series A funding, early-stage life sciences company Arbor Biotechnologies came out of stealth mode last week.
But the new firm — which was cofounded by Broad Institute scientists and CRISPR experts Winston Yan, David Scott, and Feng Zhang, as well as David Walt of Harvard University and the Wyss Institute, who also was a cofounder of Illumina and Quanterix — had something more than just a few million dollars and some nice sound bites from venture capitalists. It had a proprietary biomolecule discovery platform that had already produced results: a brand new CRISPR enzyme called Cas13d.
In a paper in Molecular Cell, researchers from Arbor and the National Institutes of Health reported on their search for novel CRISPR-Cas systems, particularly those categorized as class 2, as those systems utilize a single RNA-guided protein effector to mitigate viral infection. "We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D," the authors wrote.
They found that the median size of Cas13d proteins is 190 to 300 amino acids smaller than the various Cas13a, Cas13b, and Cas13c proteins, but that despite their small size, Cas13d orthologs from Eubacterium siraeum and Ruminococcus sp. are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage.
"The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection," the researchers added.
Indeed, the discovery of Cas13d — and the particular emphasis on its uses in RNA editing and diagnostics — would seem like a natural discovery for a group of Broad scientists to make. Zhang and his team made a splash last October when they described in Science their CRISPR-based RNA editing system, called RNA Editing for Programmable A to I Replacement (REPAIR), which allows for the temporary repair of single RNA nucleotides in mammalian cells without permanently altering the genome. The researchers profiled Type VI CRISPR systems to engineer a Cas13 ortholog capable of robust knockdown and used catalytically-inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 to transcripts in mammalian cells.
On the diagnostics side, Zhang's lab recently published the second version of its Specific High sensitivity Enzymatic Reporter unlocking — or SHERLOCK — platform, which multiplexes Cas13 with other CRISPR enzymes to detect various nucleic acids.
Google-like
But it's the way that Cas13d was discovered that Arbor is excited to tout. In a statement, the firm said its proprietary platform "employs a diverse set of technologies and techniques — including artificial intelligence, genome sequencing, gene synthesis, and high-throughput screening — to curate and mine the natural genetic diversity for impactful peptides, proteins, and enzymes" in order to "enable the high-throughput discovery and identification of enzymes that provide new protein functionalities and catalytic activities." It essentially causes a feedback loop between computational prediction and experimental testing, allowing the company to mine new proteins and characterize their functions, Arbor added.
In the same statement, Scott noted that the platform "accelerates the rate of discovery and characterization of new biomolecules by orders of magnitude," and Yan added that Arbor was nearly ready to "convert sequence data into a catalog of protein functions."
David Cheng, founding director of the company's search engine, described it in an email to GenomeWeb as almost being inspired by the way Google's search engine looks for information on the internet.
"Instead of web pages, we are building a search for proteins. Every time we update our database, we look to include more genetic data and richer features to describe underlying protein systems," he said. "Additionally, we are running proprietary high-throughput experiments, so our searches for candidate proteins are quickly paired with hard data from the lab. The pairing of our database with functional data from experiments will constantly enhance our discovery platform."
And, as natural proteins "serve a vast diversity of functions," Scott added, "we believe a catalogue growing in comprehensiveness will be an invaluable resource for unlocking new biotechnologies for improving human health and sustainability."
The team also noted that that while Cas13d is one of the earliest molecules the platform has found and characterized, it does have the potential to find proteins outside the CRISPR space, and the company intends to share those findings when appropriate.
Concurrent discovery
But the Arbor team wasn't the only one that recently developed a computational platform for biomolecule discovery. Nor, in fact, was it the only one that discovered Cas13d. On the same day Arbor published its study in Molecular Cell, the team of Patrick Hsu at the Salk Institute for Biological Studies published a study in Cell also describing the discovery of Cas13d.
Hsu and his team developed a computational program to search bacterial DNA databases for new CRISPR proteins. They aimed to find out whether there were any CRISPR proteins that existed in nature that could solve any of the problems present in CRISPR proteins that had already been discovered.
"Has evolution already solved any of the problems that we're facing in genetic engineering? You've seen in CRISPR-Cas9 a significant amount of effort gone into protein engineering, into directed evolution, into small-molecule modification," Hsu told GenomeWeb. "At the end of the day, protein engineering is a hard problem, and we wanted to see if we could harness the natural diversity of these enzymes to find a whole solution."
In order to address these issues, the Salk researchers — like the Arbor researchers — concentrated their search on class 2 CRISPR enzymes. These proteins "integrate the surveillance and defense function into a single enzyme," Hsu said, unlike class 1 enzymes like Cas3 that require multiple proteins to enact these various functions and are therefore more complicated to engineer in the lab.
With that in mind, first author Silvana Konermann said, the team built the computational search program to look for the "core feature" of any CRISPR system: the contiguous stretch of DNA that distinguishes one Cas from another called the array. And once the program found what it thought were proteins, she added, the researchers then had to cluster them into families in order to see if they matched with any families that had been previously described.
Hsu and Konermann said they and their colleagues discovered more than one novel CRISPR protein using this computational platform, but they concentrated on Cas13d because it had two particular features that they were looking for — it targets RNA and it's uniquely small. Their study showed that the Cas13d family averages 930 amino acids in length, whereas Cas9 is about 1,100 amino acids to 1,400 amino acids long, depending on subtype, Cas13a is about 1,250 amino acids long, Cas13b is about 1,150 amino acids long, and Cas13c is about 1,120 amino acids long.
They then performed a series of experiments to see which members of the Cas13d family would have an effect on human cells and what that effect would be, and teased out what they thought was one ortholog that could be particularly useful as a therapeutic for the neurodegenerative disorder frontotemporal dementia (FTD). That ortholog came from the gut bacterium Ruminococcus flavefaciens XPD3002, so they named their RNA-editing tool CasRx.
Again, the idea of using CRISPR to destroy pathogenic RNA species that cause neurodegenerative disorders isn't necessarily new. In August 2017, researchers published a study in Cell, in which they reported using a CRISPR-Cas9-based technique to visualize and eliminate pathogenic RNA species produced by microsatellite repeat expansions in DNA that cause dominantly inherited diseases such as Huntington's disease and amyotrophic lateral sclerosis.
One of the major differences here, according to Hsu, is that Cas13d's smaller size makes it much easier to package into an adeno-associated virus, and therefore more ideal for treating brain disorders. When benchmarked against RNA interference through transcriptome profiling, the researchers found that the knockdown effects of CasRx were much more specific than RNAi.
"What we're doing is taking an orthogonal system from bacteria that doesn't have crosstalk with any native processes in the cell and give it one function. So, what you see in our data is very specific knockdown," Hsu said.
Further, Hsu and Konermann noted, because Cas13d can be catalytically inactivated and converted into what Hsu called "a universal RNA-binding module," they believe it can be used as a platform for flexible transcriptome engineering and to develop tools that can go beyond simply knocking down RNA — which is largely what RNAi is limited to. They're aiming instead to develop tools that can also bind to RNAs of interest, cis elements, or pre-mRNAs to block splicing in order to tune protein isoforms or change their ratios directly inside the cell.
In order to test this function, they delivered Cas13d into a neuronal model of FTD and found that they could bring the dysregulated tau isoforms that are a hallmark of the disease back into a healthy balance, Konermann said.
The team is now conducting and planning further studies using Cas13d and CasRx. The researchers have additional models of FTD, including animal models, as well as other diseases that they can investigate using this enzyme.
"We're in the early days of figuring this out. This is a programmable RNA-targeting technology, so certainly it could be used in many different contexts, to study RNA biology in the lab or for targeting defects at the RNA level for potential therapeutic development," Hsu said. "And this could also be used for FTD, but also for many other diseases that involve RNA defects."
They also plan to continue developing the tool itself, conducting protein engineering studies on Cas13d, researching the possible uses of the other Cas13d orthologs, and, importantly, studying the potential of the other proteins their computational pipeline also found.
Thanks to all the metagenomic sequencing studies being conducted, Konermann added, there's more data on microbial genomes being released now than there ever has been before. That explains why there are still so many CRISPR enzymes left to find. And the group's computational program is being constantly fed new data.
The business of sharing
It's clear that the work on Cas13d has only just begun. Now that it has been published, its various orthologs will likely be featured in many studies in coming years for their therapeutic and diagnostic abilities. Indeed, if Hsu and Konermann's comments are anything to go by, the Salk Institute will probably be publishing a great number of those papers, itself.
But with each new CRISPR enzyme and piece of CRISPR technology comes the question of who will own the rights.
Of course, Arbor has already filed multiple provisional patents to protect the intellectual property related to its discovery platform, Cheng said. But, he also noted, "We have filed patent applications related to our work on Cas13d, which will proceed through the standard review process by the patent office."
Concurrent discoveries are not at all new in science, and they're certainly not new in the CRISPR field. All one has to do is look at the story of Cas9 to see the truth in that — the Broad Institute and the University of California, Berkeley have based their patent arguments of the past several years over who was first to discover and publish the landmark research.
But, a similar patent fight over Cas13d may not be so likely.
"On whether another patent battle is in the offing, my guess is no for two reasons. First, at this stage in the evolution of the technology, where the probability of getting patents with broad scope is low, spending money on another battle is probably not economically rational," Duke University Law Professor Arti Rai said in an email to GenomeWeb. "Second, the US system has, since March 2013, awarded priority based on a first-to-file test. That makes the answer of who is 'first' easier to determine."
It's also unlikely to be a problem for academic researchers like Hsu and his colleagues, at least for now. "We're happy to make Cas13d available for academic research labs through [the nonprofit global plasmid repository] Addgene," Cheng said. "We're also open to discussions with companies that have a commercial interest in Cas13d."
Arizona State University Professor Robert Cook-Deegan said in an email that he believes this strategy will probably be workable, at least in the short term. "It is probably true that under the framework through Addgene, 'noncommercial' research will be fine if folks use the reagents they get from Addgene. And Addgene and [Arbor] are simply not going to sue academic groups who make their own either," he said.
However, Cook-Deegan noted, the patents may come to matter in the future if academic groups develop Cas13d-based technology that they wish to commercialize. "If anyone finds any real-world use that entails use of the enzyme, then they might find themselves needing money to develop and get the tech to market. And then the patents will matter, and the job of the patent prosecutors (the folks seeking the patent rights for their clients) is to secure rights as broad and valuable as they can, which in this case would presumably have been licensed exclusively to the new startup."
For his part, Hsu commended the Arbor researchers for their work, as the Arbor researchers did the Salk team. But, he said, the discovery of Cas13d makes up only the first third of his group's new paper. "We've spent a much more significant amount of time turning it into an RNA-targeting tool that works for the community, and showing that it can be used for knock down, for splicing, to do RNA interference, to test it in disease models, and so what we're interested in is not just the discovery of new CRISPR systems, but their application," he noted.
As for Arbor, Cheng said the firm isn't looking to raise any additional funds after its Series A round last June or sign partnership deals, but rather is focused on using the platform internally for more discoveries to come. "We don't currently have plans for any wide-scale licensing of the Arbor discovery platform, although we will evaluate potential partnerships on a case-by-case basis," Cheng added.
"Just like CRISPR-Cas9 unlocked a wide range of new tools and capabilities for manipulating DNA, we think the field is just getting started with exploring the potential of the more recently discovered Cas13d," Yan said in an email. "We're really excited to introduce Cas13d as a new tool in the RNA manipulating toolbox and are looking forward to engaging with the field to maximize the positive impact it can have in the world."
Hsu and Konermann are likewise concentrating on the potential of their work.
"We're able to change the levels of RNA just by knock down, and to alter isoforms. Potentially, [CasRx] could be relevant for any disease where you have a dysregulation of transcript levels, or a toxic protein that you want to knock down, or an isoform that might be toxic, and there's really a broad range of disease where those two things apply," Konermann said. "And, this is not here to replace DNA targeting. This is here to complement. It has its own unique features that make one tool better for one application and another tool better for another application, so you're completing the tool set further."