(Nanowerk NewsA team of researchers led by Feng Zhang at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have devised the first programmable RNA guided system in eukaryotes – organisms that include fungi, plants and animals.
In a study at Natural (“Fanzor is a eukaryotic programmable RNA-guided endonuclease”), the team explains how the system is based on a protein called Fanzor. They demonstrated that the Fanzor protein uses RNA as a guide to precisely target DNA, and that Fanzor can be reprogrammed to edit the genome of human cells. The compact Fanzor system has the potential to be more easily delivered to cells and tissues as a therapy than CRISPR/Cas systems, and further refinements to improve targeting efficiency could make it a valuable new technology for human genome editing.
CRISPR/Cas was first discovered in prokaryotes (bacteria and other single-celled organisms that lack nuclei) and scientists including Zhang’s lab have long wondered if similar systems existed in eukaryotes. The new study shows that RNA-guided mechanisms of DNA cutting exist in all the kingdoms of life.
“CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome,” said Zhang, senior author of the study and member of the core institute at Broad, a researcher at MIT’s McGovern Institute, the James Professor of Neuroscience and Patricia Poitras at MIT, and Howard Hughes Medical Institute investigator. “This new system is another way to make precise changes to human cells, complementing the genome editing tools we already have.”
Searching for the domain of life
Zhang’s lab’s ultimate goal is to develop genetic drugs using systems that can modulate human cells by targeting specific genes and processes. “A few years ago, we started asking, ‘What lies beyond CRISPR, and are there other RNA-programmable systems in nature?’” says Zhang.
Two years ago, members of Zhang’s lab discovered a class of programmable RNA systems in prokaryotes called OMEGA (Science, “The widely distributed IS200/IS605 transposon family encodes a variety of programmable RNA-guided endonucleases”), which are often associated with transposable elements, or “jumping genes”, in the bacterial genome and possibly give rise to the CRISPR/Cas system. The work also highlighted similarities between the prokaryotic OMEGA system and Fanzor proteins in eukaryotes, suggesting that Fanzor enzymes might also use RNA-guided mechanisms to target and cut DNA.
In the new study, the researchers continued their study of RNA-guided systems by isolating Fanzors from a species of fungus, algae, and amoeba, as well as a clam known as the Northern Quahog. Co-first author Makoto Saito from Zhang’s lab led the biochemical characterization of Fanzor proteins, showing that they are DNA-cutting endonuclease enzymes that use nearby non-coding RNAs known as ωRNAs to target specific sites in the genome. This is the first time this mechanism has been discovered in eukaryotes, such as animals.
Unlike CRISPR proteins, Fanzor enzymes are encoded in eukaryotic genomes in transposable elements and the team’s phylogenetic analysis showed that Fanzor genes had migrated from bacteria to eukaryotes via what is called horizontal gene transfer.
“The OMEGA system is more of an ancestor of CRISPR and is one of the most abundant proteins on the planet, so it makes sense that they would be able to jump back and forth between prokaryotes and eukaryotes,” said Saito.
To explore Fanzor’s potential as a genome editing tool, the researchers demonstrated that it can generate insertions and deletions at targeted genomic sites in human cells. The researchers found that the Fanzor system was initially less efficient at cutting DNA than the CRISPR/Cas system, but by systematic engineering, they introduced a combination of mutations into the protein that increased its activity 10-fold. In addition, unlike some CRISPR systems and the OMEGA TnpB protein, the team found that the mushroom-derived Fanzor protein does not exhibit “collateral activity,” in which an RNA-guided enzyme cleaves its target DNA as well as degrades nearby DNA or RNA. The results show that Fanzors has the potential to be developed as an efficient genome editor.
First co-author Peiyu Xu led an effort to analyze the molecular structure of the Fanzor/ωRNA complex and illustrate how it attaches to DNA to cut it. Fanzor shares structural similarities with its prokaryotic partner CRISPR-Cas12 protein, but the interaction between ωRNA and Fanzor’s catalytic domain is more extensive, suggesting that ωRNA may play a role in the catalytic reaction. “We are excited about this structural insight to help us further design and optimize Fanzor to increase efficiency and precision as a genome editor,” said Xu.
Like CRISPR-based systems, the Fanzor system can be easily reprogrammed to target specific genomic sites, and Zhang says it could one day be developed into a powerful new genome-editing technology for research and therapeutic applications. The abundance of RNA-guided endonucleases such as Fanzors further expands the number of known OMEGA systems across the kingdom of life and indicates that there is still much more to be discovered.
“Nature is amazing. There is so much diversity,” said Zhang. “There may be more RNA-programmable systems out there, and we continue to explore and hopefully find many more.”