Scientists, typically, are on their own after their post-doc ends. Sometimes, albeit rarely, they team up, blending their ideas, money, and people. Connie Cepko, PhD, and Cliff Tabin, PhD, pioneered the idea of a lab partnership, spending the last few decades working together in their shared lab at Harvard Medical School. Elçin Ünal and Gloria Brar adopted this team approach when they started their lab together (the Br-Ün lab) at University of California at Berkeley in 2014. And, in the spring of 2019, Omar Abudayyeh, PhD, and Jonathan Gootenberg, PhD, followed suit, setting up their joint lab at the McGovern Institute at MIT. They decided on this path—and passed on doing a traditional post-doc—in large part so that they could keep working together. Why? “We work well as a team,” says Abudayyeh.
In just two years, their partnership has paid off. In their latest paper, the lab describes Cas7-11, a single protein that consolidates the domains of more complex CRISPR systems. The new system is akin to a type III CRISPR system, which have many subunits and form large complexes, making them difficult to use as tools. The Cas7-11 system, however, has elegantly condensed all the domains into a single protein that cuts RNA.
The work is published in Nature in the paper, “Programmable RNA targeting with the single-protein CRISPR effector Cas7-11.”
Unlike Cas13, an RNA targeting CRISPR system previously discovered by Abudayyeh and Gootenberg during their graduate work in the lab of Feng Zhang, PhD, professor at MIT, Cas7-11 does not result in collateral cutting. Cas13, once activated, cuts other RNA in the cell. This property eliminates it from the list of candidates that could have utility in a therapeutic capacity in vivo. In a stroke of luck for Abudayyeh and Gootenberg, Cas7-11 doesn’t have that same activity; it cuts its target RNA and nothing more.
Beginning with evolutionary biologists
Abudayyeh and Gootenberg’s paper was launched by the work of Kira Makarova, PhD, staff scientist in the lab of Eugene Koonin, PhD, distinguished investigator in the evolutionary genomics research group at the NIH. Her initial identification of CRISPR-subtype III-E, with its single-protein effector, was the discovery that sits at the cornerstone of the project.
Joe Peters, PhD, professor at Cornell University, explains that genomes undergo random insertions and deletions, producing meaningless combinations as gene sets recombine next to one another. While the vast majority have no functionality, there are the needles-in-the-haystack that produce functional combinations of genes. The very careful work required to uncover them often involves identifying the phylogenetic signature that indicates that it is not a chance association. This is very hard to do notes Peters, adding that Makarova is one of the few people with the experience and talent to pull it off.
When Abudayyeh and Gootenberg heard Makarova present her data at a conference, specifically that that the pieces of the CRISPR system had fused together into one protein, their interest was piqued.
The significance of the work in the paper is twofold, notes Koonin. First, subtype III-E (Cas7-11) reveals a novel route of CRISPR evolution, via fusion of effector complex subunits accompanied by loss of several components. Under this scenario, a single-protein CRISPR effector resembling Class II effectors (Cas9, Cas12, Cas13) evolved from a typical, multisubunit Class I effector. This is a completely unexpected path of evolution, he notes.
Second, Koonin points to the fact that Cas7-11 has lost the capacity to couple immunity with cell death. Meaning that it can cut RNA while leaving the cell alive. This is biologically interesting, he notes, and provides potential major advantages for developing tools for RNA manipulation.
A class I system made up of one fusion protein was not only an interesting evolutionary finding, it was potentially a very useful tool.
Did Koonin know that this system would turn out to be so useful? “We did not know,” he explains, “but it was possible to speculate given the loss of the signaling component required for coupling of immunity to programed cell death.”
The team does a believable job, adds Peters, of assembling clues together for how a system that was ancestrally multiple parts (a classic class I system) evolved into a single protein effector (like what one would expect for a class II system) capable of processing the guide and cleaving the target mRNA. The paper, he adds, presents another example, where detailed phylogenic studies aren’t just a wonks pursuit, but one that can lead to useful tools.
The Cas9 for RNA?
The purified complex was first published a month ago in Science, by a team at the Kavli Institute of Nanoscience in Delft, Netherlands. In that paper, the Brouns lab showed that the complex targeted and cut RNA. But this current paper tells much more of the story.
The team from the “AbuGoot” lab presents in vivo work in both bacteria and mammalian cells. The lion’s share of the work, according to Gootenberg, was the engineering to bring the protein from bacteria to human cells. This includes, but is not limited to, codon optimization, targeting through nuclear localization sequences, export sequences, scaffolds for expressing the guide with direct repeat sequences, and “tinkering around with pieces” that lead to increases in efficiency and potency. These engineering efforts, Gootenberg adds, are never really completed and the lab will continue to work on optimizing the system.
Although this system, and the field, is being categorized as RNA editing, the potential goes far beyond just changing a base pair. Cas7-11 could be used to generate RNA knockdowns, RNA stabilization which results in higher levels of protein, and RNA base editing.
RNA editing is a concise, and limited, term, notes Abuddayah, who prefers the term “transcriptome engineering.” He can imagine this system being used to recruit proteins and domains to facilitate epitranscriptomic modulation, splicing modulation, and more. “The list goes on,” he notes.
Cas7-11 does not fit into an AAV vector, a stumbling block toward the goal of therapeutic applications in humans. One potential workaround came in the discovery of a smaller variant (with the same mechanism and function) hvsCas7-11. hvsCas7-11 can be packaged into two AAVs (one for the guide and one for the protein.) The lab will continue to optimize this process, says Gootenberg. “There are a lot ways to shrink an enzyme,” he adds.
In addition to Sherlock Biosciences, a company that develops diagnostic tools using Cas13, Abudayyeh and Gootenberg are involved in three other companies: a CRISPR‑based, COVID‑19 molecular testing company named Proof Diagnostics, Moment Biosciences which, according to an online video, sits at the intersection of genetic engineering, the microbiome and inflammatory disease, and Tome Biosciences—incorporated earlier this year.
There is a wide array of diseases, Gootenberg says, for which DNA-based therapeutics are not the best option. Abuddayah mentions that Cas7-11 goes beyond making permanent changes and allows for more temporal modulation because it affects RNA. He mentions the modulation of pain receptors, inflammation, and infectious diseases such as SARS-CoV-2 and other RNA viruses. Although not ready to talk about how Cas7-11 may play into the future of human disease, the pair seem ready to play in the drug development game.
