An unlimited search of natural diversity has led scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute of MIT and Harvard to uncover ancient systems with potential to expand the genome editing toolbox.
These systems, which the researchers call TIGR (Tandem Interspaced Guide RNA) systems, use RNA to guide them to specific sites on DNA. TIGR systems will be reprogrammed to focus on any DNA sequence of interest, and so they have distinct functional modules that may act on the targeted DNA. Along with its modularity, TIGR could be very compact in comparison with other RNA-guided systems, like CRISPR, which is a significant advantage for delivering it in a therapeutic context.
These findings are reported online Feb. 27 within the journal .
“This can be a very versatile RNA-guided system with numerous diverse functionalities,” says Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT, who led the research. The TIGR-associated (Tas) proteins that Zhang’s team found share a characteristic RNA-binding component that interacts with an RNA guide that directs it to a selected site within the genome. Some cut the DNA at that site, using an adjoining DNA-cutting segment of the protein. That modularity could facilitate tool development, allowing researchers to swap useful recent features into natural Tas proteins.
“Nature is pretty incredible,” says Zhang, who can also be an investigator on the McGovern Institute and the Howard Hughes Medical Institute, a core member of the Broad Institute, a professor of brain and cognitive sciences and biological engineering at MIT, and co-director of the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics at MIT. “It’s got an incredible amount of diversity, and we’ve got been exploring that natural diversity to search out recent biological mechanisms and harnessing them for various applications to govern biological processes,” he says. Previously, Zhang’s team adapted bacterial CRISPR systems into gene editing tools which have transformed modern biology. His team has also found a wide range of programmable proteins, each from CRISPR systems and beyond.
Of their recent work, to search out novel programmable systems, the team began by zeroing in a structural feature of the CRISPR-Cas9 protein that binds to the enzyme’s RNA guide. That may be a key feature that has made Cas9 such a strong tool: “Being RNA-guided makes it relatively easy to reprogram, because we understand how RNA binds to other DNA or other RNA,” Zhang explains. His team searched tons of of hundreds of thousands of biological proteins with known or predicted structures, searching for any that shared the same domain. To seek out more distantly related proteins, they used an iterative process: from Cas9, they identified a protein called IS110, which had previously been shown by others to bind RNA. They then zeroed in on the structural features of IS110 that enable RNA binding and repeated their search.
At this point, the search had turned up so many distantly related proteins that they team turned to artificial intelligence to make sense of the list. “If you end up doing iterative, deep mining, the resulting hits will be so diverse that they’re difficult to investigate using standard phylogenetic methods, which depend on conserved sequence,” explains Guilhem Faure, a computational biologist in Zhang’s lab. With a protein large language model, the team was capable of cluster the proteins that they had found into groups in response to their likely evolutionary relationships. One group set other than the remainder, and its members were particularly intriguing because they were encoded by genes with repeatedly spaced repetitive sequences paying homage to an integral part of CRISPR systems. These were the TIGR-Tas systems.
Zhang’s team discovered greater than 20,000 different Tas proteins, mostly occurring in bacteria-infecting viruses. Sequences inside each gene’s repetitive region — its TIGR arrays — encode an RNA guide that interacts with the RNA-binding a part of the protein. In some, the RNA-binding region is adjoining to a DNA-cutting a part of the protein. Others appear to bind to other proteins, which suggests they could help direct those proteins to DNA targets.
Zhang and his team experimented with dozens of Tas proteins, demonstrating that some will be programmed to make targeted cuts to DNA in human cells. As they consider developing TIGR-Tas systems into programmable tools, the researchers are encouraged by features that would make those tools particularly flexible and precise.
They note that CRISPR systems can only be directed to segments of DNA which can be flanked by short motifs often called PAMs (protospacer adjoining motifs). TIGR Tas proteins, in contrast, don’t have any such requirement. “This implies theoretically, any site within the genome needs to be targetable,” says scientific advisor Rhiannon Macrae. The team’s experiments also show that TIGR systems have what Faure calls a “dual-guide system,” interacting with each strands of the DNA double helix to home in on their goal sequences, which should ensure they act only where they’re directed by their RNA guide. What’s more, Tas proteins are compact — 1 / 4 of the dimensions Cas9, on average — making them easier to deliver, which could overcome a significant obstacle to therapeutic deployment of gene editing tools.
Excited by their discovery, Zhang’s team is now investigating the natural role of TIGR systems in viruses, in addition to how they will be adapted for research or therapeutics. They’ve determined the molecular structure of one in every of the Tas proteins they found to work in human cells, and can use that information to guide their efforts to make it more efficient. Moreover, they note connections between TIGR-Tas systems and certain RNA-processing proteins in human cells. “I believe there’s more there to check by way of what a few of those relationships could also be, and it could help us higher understand how these systems are utilized in humans,” Zhang says.
This work was supported by the Helen Hay Whitney Foundation, Howard Hughes Medical Institute, K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics, Broad Institute Programmable Therapeutics Gift Donors, Pershing Square Foundation, William Ackman, Neri Oxman, the Phillips family, J. and P. Poitras, and the BT Charitable Foundation.
