Programmable RNA-guided DNA nucleases play various roles in prokaryotes, but how widely they have expanded outside of these organisms is still being determined. The activity and functions of factors, the eukaryotic homolog of bacterial TnpB proteins, have been discovered in the genomes of eukaryotes and big viruses.
A new study from MIT‘s McGovern Institute for Brain Research has identified thousands of Fanzors in diverse species, from snails to algae to amoebas. The newly recognized diversity of natural Fanzor enzymes gives scientists an extensive set of programmable enzymes that might be adapted into new tools for research or medicine.
CRISPR-based genome editing tools have helped scientists uncover other RNA-guide enzymes throughout the bacterial world, many with features that make them valuable in the lab. An entirely new area of RNA-guided biology has been opened by the discovery of Fanzors, whose capacity to cut DNA in an RNA-guided manner.
The eukaryotic organisms, which include plants, animals, and fungi, are characterized by the membrane-bound nucleus that houses each cell’s genetic material. Fanzors were the first such enzymes to be discovered in eukaryotic species.
Enzymes that naturally evolved in eukaryotic organisms might be better suited to function safely and efficiently in the cells of other eukaryotic organisms, including humans. Scientists have shown that Fanzor enzymes can be engineered to cut specific DNA sequences in human cells precisely.
In this study, scientists discovered that some Fanzors can target DNA sequences in human cells without optimization. Seeing that they operate pretty effectively in mammalian cells was terrific.
Numerous Fanzors had been discovered among eukaryotic creatures before this study. An order of magnitude has now increased the known diversity of these enzymes through a thorough search of genetic databases.
Five distinct families of the enzymes could be distinguished among the more than 3,600 Fanzors discovered in eukaryotes and the viruses that infect them. The specific nature of these enzymes was compared, and they found proof of a lengthy evolutionary history.
Fanzors most likely originated from TnpBs, RNA-guided DNA-cutting enzymes found in bacteria. Scientists were first interested in the genetic similarities between Fanzors and these bacterial enzymes.
The evolutionary linkages discovered by Gootenberg and Abudayyeh imply that the bacterial ancestors of Fanzors likely infiltrated eukaryotic cells more than once, starting their evolution. Viruses probably spread some of these, while symbiotic bacteria might have brought others about. The research also implies that the enzymes evolved properties appropriate for their new habitat after being incorporated by eukaryotes, such as a signal that permits them to enter a cell nucleus where they can access DNA.
Scientists conducted genetic and biochemical experiments. They found that Fanzors have evolved a DNA-cutting active site distinct from their bacterial predecessors.
The ancestors of TnpB, when addressed to a stretch of DNA in a test tube, get activated and cut other sequences in the box; Fanzors lack this promiscuous activity, allowing the enzyme to miss its target sequence more precisely. They discovered that particular Fanzors could cut these target sequences with roughly 10 to 20 percent effectiveness when they utilized an RNA guide to direct the enzymes to cut specific locations in the genome of human cells.
McGovern Fellow Jonathan Gootenberg said, “With further research, Abudayyeh and Gootenberg hope that various sophisticated genome editing tools can be developed from Fanzors. “It’s a new platform, and they have many capabilities.”
McGovern Fellow Omar Abudayyeh said, “Opening up the whole eukaryotic world to these types of RNA-guided systems is going to give us much to work on.”
Journal Reference:
- Kaiyi Jiang, Justin Lim, Samantha Sgrizzi et al. Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses. Science Advances. DOI: 10.1126/sciadv.adk0171