MIT researchers have discovered a large number of programmable DNA-cutting enzyme Fanzors from eukaryotes, expanding the gene-editing potential of RNA-guided tools and opening up the possibility for more precise and efficient genome modification, especially in human cells.
New research finds that RNA-guided enzymes called Fanzors are widespread in eukaryotes. A wide variety of species, from snails to algae to amoebas, make programmable DNA-cutting enzymes called Fanzors - and a new study by scientists at MIT's McGovern Institute for Brain Research has discovered thousands of such enzymes.
Fanzors are RNA-guided enzymes that can be programmed to cut DNA at specific sites, much like bacterial enzymes, and power the widely used gene-editing system CRISPR. Recently, the journal Science Advances reported the newly discovered diversity of natural Fanzor enzymes, providing scientists with a broad set of programmable enzymes that could potentially be engineered into new research or medical tools.
"RNA-guided biology allows you to make programmable tools that are really easy to use. So the more we can find, the better," said McGovern Fellow Omar Abudayeh, who co-led the study with McGovern Fellow Jonathan Guttenberg.
CRISPR is an ancient bacterial defense system that clearly demonstrates the utility of RNA-guided enzymes in the laboratory. The CRISPR-based genome editing tools developed by MIT professors and McGovern Fellows Zhang Feng, Abudaye, Guttenberg and others have changed the way scientists modify DNA, accelerated the research process, and contributed to the development of many experimental gene therapies.
Researchers have since discovered other RNA-guided enzymes in the bacterial world, many of which have properties that make them valuable in the laboratory. Zhang's group reported earlier this year the discovery of Fanzors, which can cut DNA in an RNA-guided manner, opening up a new field of RNA-guided biology.
Fanzors are the first enzymes of this type discovered in eukaryotes. Eukaryotes are a large group of life forms including plants, animals and fungi that are defined by a membrane-bound nucleus that holds each cell's genetic material. (Bacteria without a nucleus are prokaryotes).
"People have been looking for interesting tools in prokaryotic systems for a long time, and I think this has yielded incredible results," Gutenberg said. "Eukaryotic systems are really a whole new place to work."
One hope is that enzymes that evolved naturally in eukaryotes might be better suited to function safely and efficiently in the cells of other eukaryotes, including humans. The research team has demonstrated that the Fanzor enzyme can precisely cut specific DNA sequences in human cells.
In the new study, Abudayeh and Guttenberg found that some Fanzors can target DNA sequences in human cells even if they are not optimized. They work surprisingly efficiently in mammalian cells.
Prior to this study, hundreds of Fanzors had been discovered in eukaryotes. Through extensive searches of genetic databases, led by lab member Justin Lim, Guttenberg and Abudaye's team has now expanded the known diversity of these enzymes by an order of magnitude.
Among the more than 3,600 Fanzors found in eukaryotes and viruses that infect eukaryotes, the researchers were able to identify five distinct families of enzymes. By comparing the precise makeup of these enzymes, they found evidence of a long evolutionary history.
Fanzors likely evolved from RNA-guided DNA-cutting bacterial enzymes called TnpBs. In fact, it was Fanzors' genetic similarity to these bacterial enzymes that first attracted the attention of Zhang's research group and the teams of Guttenberg and Abudaye.
The evolutionary connections the researchers traced suggest that these bacterial precursors to Fanzors likely entered eukaryotic cells more than once to kick-start their evolution. Some of these may be transmitted by viruses, while others may be introduced by commensal bacteria. The research also shows that after being taken up by eukaryotes, these enzymes evolved characteristics adapted to their new environment, such as signals that allowed them to enter the cell nucleus, where they could access DNA.
Through genetic and biochemical experiments led by bioengineering graduate student Kaiyi Jiang, the team determined that Fanzors have evolved a DNA-cleaving active site that is distinct from the active site of their bacterial predecessors. When TnpB's ancestor targets a DNA sequence in the test tube, it becomes activated and cuts other sequences in the test tube; Fanzors lack this disorganized activity. When they used an RNA guide to guide the enzyme to cut specific sites in the genome of human cells, they found that certain Fanzors were able to cut these target sequences with about 10 to 20 percent efficiency.
As their research progresses, Abudayeh and Guttenberg hope to use Fanzors to develop a variety of sophisticated genome-editing tools: "This is a new platform, and they have a lot of capabilities. Opening up the entire eukaryote community to these types of RNA-guided systems will give us a lot of research opportunities."