Thousands of programmable DNA-cutters found in algae, snails, and other organisms

Thousands of different species, including snails, algae, and amoebas, are known to produce programmable DNA-cutting enzymes known as Fanzors. These species have been discovered in a recent study by researchers at MIT's McGovern Institute for Brain Research. Similar to the bacterial enzymes that drive the popular CRISPR gene-editing system, fanzors are RNA-guided enzymes that may be designed to cut DNA at particular locations. Scientists now have a large repertoire of programmable enzymes to choose from when creating novel research or therapeutic tools thanks to the recently identified variety of natural Fanzor enzymes, which was published on September 27 in the journal Science Advances.

"RNA-guided biology is the key to creating very user-friendly programmed technologies. Thus, the more information we can get, the better, says Omar Abudayyeh, a McGovern Fellow who oversaw the study alongside Jonathan Gootenberg.

An old bacterial defensive mechanism called CRISPR has demonstrated the utility of RNA-guided enzymes when modified for laboratory usage. The method scientists alter DNA has altered as a result of the CRISPR-based genome editing techniques created by MIT professor and McGovern investigator Feng Zhang, Abudayyeh, Gootenberg, and others. This has sped up research and made it possible to generate several experimental gene treatments.

Since then, other RNA-guide enzymes have been found in various bacterial species by researchers, several of which have characteristics that make them useful in the laboratory. An exciting new area of RNA-guided biology has been revealed by the discovery of Fanzors, whose capacity to cut DNA in an RNA-guided fashion was reported by Zhang's lab earlier this year. The first of these enzymes to be discovered in eukaryotic creatures—a broad category of living species that includes plants, animals, and fungi—were fanzors. These organisms are distinguished by their membrane-bound nuclei, which house the genetic material of each cell. (Bacteria are members of the prokaryotes, a group that does not have a nucleus.)

"People have been searching for interesting tools in prokaryotic systems for a long time, and I think that that has been incredibly fruitful," Gootenberg adds. "Eukaryotic systems are really just a whole new kind of playground to work in."

According to Abudayyeh and Gootenberg, there is a chance that enzymes that have developed spontaneously in eukaryotic creatures will be more adapted to operate effectively and safely in the cells of other eukaryotic organisms, such as humans. Fanzor enzymes can be created to precisely cut certain DNA sequences in human cells, as Zhang's group has demonstrated. Abudayyeh and Gootenberg found in their latest research that some Fanzors can target human cell DNA sequences even in the absence of optimization. "The fact that they work quite efficiently in mammalian cells was really fantastic to see," Gootenberg explains.

Hundreds of Fanzors have been discovered among eukaryotic creatures prior to the current investigation. As a result of Gootenberg and Abudayyeh's team doing a thorough search of genetic databases under the direction of lab member Justin Lim, the known variety of these enzymes has now increased significantly.

The researchers identified five distinct families of enzymes among the more than 3,600 Fanzors they discovered in eukaryotes and the viruses that infect them. Through detailed compositional comparison, scientists discovered indications of a protracted evolutionary past.

Fanzors most likely descended from TnpBs, RNA-guided bacterial enzymes that cleave DNA. In fact, Zhang's group and Gootenberg and Abudayyeh's team were initially drawn to Fanzors because of their genetic resemblance to these bacterial enzymes.

Gootenberg and Abudayyeh's evolutionary linkages imply that these Fanzor-ancestors most likely invaded eukaryotic cells more than once, starting their evolution. Some could have been brought in by symbiotic bacteria, while others were probably spread by viruses. Additionally, the study implies that the enzymes acquired adaptations to adapt to their new home in eukaryotes, including a signal that enables them to enter a cell nucleus and access DNA.

Under the direction of graduate student Kaiyi Jiang in biological engineering, the team conducted genetic and biochemical studies that revealed that Fanzors had evolved a DNA-cutting active site that is different from that of their bacterial ancestors. The ancestors of TnpB, when targeted to a DNA sequence in a test tube, get active and cut other sequences in the tube; Fanzors lack this promiscuous activity. This appears to allow the enzyme to cut its target sequence more accurately. They discovered that some Fanzors could cut these target sequences with an efficiency of around 10–20% when they employed an RNA guide to instruct the enzymes to cut particular locations in the human cell genome.

Abudayyeh and Gootenberg want to construct a range of advanced genome editing tools from Fanzors with more study. Gootenberg states, "It's a new platform, and they have many capabilities."

"Opening up the whole eukaryotic world to these types of RNA-guided systems is going to give us a lot to work on," says Abudayyeh.