Researchers Discover Programmable RNA-Guided System in Eukaryotes

Scientists led by Feng Zhang at the Broad Institute of MIT and Harvard, have identified the first programmable RNA-guided system in eukaryotes. Published in Nature, the study reveals that this system, based on a protein called Fanzor, utilizes RNA as a guide to accurately target DNA, thereby allowing the editing of the human genome. The compact nature of the Fanzor system presents a potential advantage in delivering therapeutics to cells and tissues compared to the widely used CRISPR/Cas systems. Further improvements to enhance its targeting efficiency could establish Fanzor as a valuable technology for human genome editing.

While CRISPR/Cas systems were originally found in prokaryotes, scientists have long wondered if similar systems exist in eukaryotes. This study demonstrates the presence of RNA-guided DNA-cutting mechanisms across all kingdoms of life.

Zhang, the senior author of the study, explains that CRISPR-based systems are powerful because of their versatility in targeting different sites in the genome. The discovery of the Fanzor system provides an additional means of making precise changes in human cells, complementing the existing genome editing tools.

The researchers conducted an extensive search for RNA-programmable systems beyond CRISPR. Two years ago, they identified a class of RNA-programmable systems in prokaryotes called OMEGAs, which are associated with transposable elements in bacterial genomes and likely served as the precursor to CRISPR/Cas systems. This earlier work revealed similarities between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that Fanzor enzymes might also employ an RNA-guided mechanism to target and edit DNA.

In the current study, the researchers isolated Fanzors from various eukaryotes, including fungi, algae, amoeba species, and a clam called the Northern Quahog. Biochemical characterization conducted by the Zhang lab demonstrated that Fanzor proteins are DNA-cutting endonucleases that utilize ωRNAs (nearby non-coding RNAs) to target specific sites in the genome. This mechanism, previously unidentified in eukaryotes, offers new insights into animal genetics.

Unlike CRISPR proteins, Fanzor enzymes are encoded in the eukaryotic genome within transposable elements. Phylogenetic analysis suggests that Fanzor genes migrated from bacteria to eukaryotes through horizontal gene transfer.

To assess the potential of Fanzor as a genome editing tool, the researchers successfully demonstrated its ability to generate insertions and deletions at targeted genome sites in human cells. Although the initial efficiency of the Fanzor system in cleaving DNA was lower than CRISPR/Cas systems, systematic engineering led to the introduction of mutations that increased its activity by 10-fold. Moreover, unlike certain CRISPR systems, a fungal-derived Fanzor protein did not exhibit "collateral activity" by degrading nearby DNA or RNA. These findings indicate that Fanzors have the potential to be developed into efficient genome editors.

The researchers also analyzed the molecular structure of the Fanzor/ωRNA complex, discovering similarities to the prokaryotic CRISPR-Cas12 protein. However, the interaction between ωRNA and the catalytic domains of Fanzor was found to be more extensive, suggesting the ωRNA's involvement in the catalytic reactions. This structural insight offers a foundation for further engineering and optimization of Fanzor to enhance its efficiency and precision as a genome editor.