Researchers at MIT’s McGovern Institute and the Broad Institute of MIT and Harvard have discovered an RNA-guided system, dubbed TIGR (Tandem Interspaced Guide RNA), that could provide a more versatile and compact tool for gene editing, which could even overcome some limitations of CRISPR-based gene editing systems. The investigators noted, in a report published in Science, that TIGR is very compact compared with other gene editing systems, including CRISPR, which could be an advantage for using it as a therapeutic gene editing tool.
“This is a very versatile RNA-guided system with a lot of diverse functionalities,” said senior author Feng Zhang, PhD, a professor of biological engineering at MIT who has done extensive work developing CRISPR gene editing systems.
The new discovery came about via a comprehensive search of natural diversity with the hope of finding ancient systems that could be used to expand the gene editing playing field.
“Nature is pretty incredible. It’s got a tremendous amount of diversity, and we have been exploring that natural diversity to find new biological mechanisms and harnessing them for different applications to manipulate biological processes,” Zhang added.
The TIGR-associated (Tas) proteins found by the researchers share a characteristic RNA-binding component. This component interacts with an RNA guide that directs it to a specific site in the genome. Some cut the DNA at that site, using an adjacent DNA-cutting segment of the protein. The team believes this modularity could facilitate swapping features into the naturally occurring Tas proteins.
Further, unlike CRISPR, which requires specific short DNA motifs, known as PAMs (protospacer adjacent motifs), to target DNA, TIGR proteins do not have this limitation. “This means theoretically, any site in the genome should be targetable,” said Rhiannon Macrae, PhD, a scientific advisor at the Broad Institute.
In their search of ancient natural biological systems, the team homed in on proteins that interact with RNA guides, a key feature of the CRISPR system. Zhang’s team focused on the RNA-binding components of proteins like Cas9, which have been pivotal in gene editing tools. Using advanced computational methods, the team identified more than 20,000 different TIGR-associated proteins, mostly found in viruses that infect bacteria. Some of these proteins proved capable of making precise cuts in DNA within human cells.
In their experiments, the team demonstrated that some TIGR proteins could be reprogrammed to target specific sites in the DNA of human cells. These proteins are not only about a quarter of the size of the Cas9 protein they are also modular. These features could lead to easier engineering and development of new gene-editing tools and their size could make them easier to deliver into cells than Cas9 proteins.
The TIGR system also has a unique dual-guide mechanism, which allows it to interact with both strands of the DNA double helix, enhancing the precision of the gene-editing process, the investigators noted.
“The ability to target both strands ensures that they act only where they are directed by their RNA guide,” said the study’s first author Guilhem Faure, PhD, a computational biologist and senior group leader in the Zhang lab. This dual-guide mechanism could make TIGR systems a more reliable tool for genetic modifications, minimizing the off-target effects that have often hampered CRISPR technology.
The researchers are now focused on understanding the natural role of TIGR systems in viruses and how these systems can be further adapted for research and therapeutic uses. “I think there’s more there to study in terms of what some of those relationships may be, and it may help us better understand how these systems are used in humans,” Zhang said.
To that end, the team has already mapped the molecular structure of one of the identified Tas proteins that they demonstrated works effectively in human cells. This structural information will guide efforts to enhance the protein’s efficiency and potentially bring it closer to real-world applications.
