Another potentially game-changing discovery has come out of the lab of Feng Zhang, PhD. This time, they uncovered the first programmable RNA-guided system in eukaryotes. Just a few months ago, the Zhang lab adapted a contractile injection system, found naturally in bacteria, that deliver protein payloads to target human cells.
In this recent work, Zhang’s team describes how the system—based on the Fanzor protein—uses RNA as a guide to target DNA precisely, and that Fanzor can be reprogrammed to edit the genome of human cells. Additionally, the compact Fanzor systems have the potential to be more easily delivered to cells and tissues as therapeutics than CRISPR/Cas systems, and further refinements to improve their targeting efficiency could make them a valuable new technology for human genome editing.
The research is published in Nature, in the paper, “Fanzor is a eukaryotic programmable RNA-guided endonuclease.”
People have long wondered whether systems similar to the well-known bacterial CRISPR system exist in eukaryotes. The new study demonstrates that RNA-guided DNA-cutting mechanisms are present across all 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, a core institute member at the Broad. “This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.”
A major aim of the Zhang lab is to develop genetic medicines using systems that can modulate human cells by targeting specific genes and processes. “A number of years ago, we started to ask, ‘What is there beyond CRISPR, and are there other RNA-programmable systems out there in nature?’” said Zhang.
Two years ago, Zhang lab members discovered a class of RNA-programmable systems in prokaryotes called OMEGAs, which are often linked with transposable elements in bacterial genomes and likely gave rise to CRISPR/Cas systems. That work also highlighted similarities between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that the Fanzor enzymes might also use an RNA-guided mechanism to target and cut DNA.
In the new study, the researchers continued their study of RNA-guided systems by isolating Fanzor from fungi, algae, and amoeba species, in addition to a clam—the Northern Quahog.
The biochemical characterization of the Fanzor proteins showed that they are DNA-cutting endonuclease enzymes that use nearby noncoding RNAs (ωRNAs) to target particular sites in the genome. It is the first time this mechanism has been found in eukaryotes.
Unlike CRISPR proteins, Fanzor enzymes are encoded in the eukaryotic genome within transposable elements and the team’s phylogenetic analysis suggests that the Fanzor genes have migrated from bacteria to eukaryotes through horizontal gene transfer.
The researchers then demonstrated that Fanzor can generate insertions and deletions at targeted genome sites within human cells. The researchers found the Fanzor system to initially be less efficient at snipping DNA than CRISPR/Cas systems, but genetically engineered the protein to increase its activity 10-fold. Additionally, unlike some CRISPR systems and the OMEGA protein TnpB, the team found that a fungal-derived Fanzor protein did not exhibit “collateral activity,” where an RNA-guided enzyme cleaves its DNA target as well as degrading nearby DNA or RNA. The results suggest that Fanzor could potentially be developed as efficient genome editors.
The group also analyzed the molecular structure of the Fanzor/ωRNA complex. They found that Fanzor shares structural similarities with CRISPR-Cas12, but the interaction between the ωRNA and the catalytic domains of Fanzor is more extensive, suggesting that the ωRNA might play a role in the catalytic reactions.
Like CRISPR-based systems, the Fanzor system can be easily reprogrammed to target specific genome sites, and Zhang said it could one day be developed into a powerful new genome editing technology for research and therapeutic applications. The abundance of RNA-guided endonucleases like Fanzor further expands the number of OMEGA systems known across kingdoms of life and suggests that there are more yet to be found.
“Nature is amazing. There’s so much diversity,” said Zhang. “There are probably more RNA-programmable systems out there, and we’re continuing to explore and will hopefully discover more.”
