First programmable RNA-guided system in eukaryotes discovered

A group of scientists led by Feng Zhang at the Broad Institute of MIT and Harvard, along with the McGovern Institute for Brain Research at MIT, has made an exciting discovery regarding programmable RNA-guided systems in eukaryotes. These organisms encompass a wide range of life forms such as fungi, plants, and animals.

Their findings, published in the journal Nature, focus on a protein called Fanzor. The researchers demonstrated that Fanzor proteins utilize RNA as a guide to accurately target DNA, and they were able to reprogram Fanzors to edit the genome of human cells. The compact nature of Fanzor systems holds promise for easier delivery to cells and tissues as therapeutics, potentially surpassing the challenges associated with CRISPR/Cas systems. By enhancing their targeting efficiency, these Fanzor systems could become a valuable tool for editing the human genome.

Although CRISPR/Cas was initially discovered in prokaryotes (single-celled organisms like bacteria lacking nuclei), scientists, including Zhang’s team, have long speculated about the existence of similar systems in eukaryotes. This new study confirms the presence of RNA-guided DNA-cutting mechanisms across all kingdoms of life.

Zhang, the senior author of the study and a core member of the Broad Institute, an investigator at MIT’s McGovern Institute, the James and Patricia Poitras Professor of Neuroscience at MIT, and a Howard Hughes Medical Institute investigator, commented on the significance of the research: “CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome. This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.”

Searching the domains of life

The primary objective of the Zhang laboratory is to develop genetic medicines utilizing systems capable of modulating human cells through targeted gene manipulation and processes. Zhang expressed their curiosity about exploring alternatives to CRISPR and the existence of other RNA-programmable systems in nature.

A couple of years ago, researchers in the Zhang lab identified a class of RNA-programmable systems called OMEGAs in prokaryotes. These systems are often associated with transposable elements or “jumping genes” in bacterial genomes and are believed to have given rise to CRISPR/Cas systems. Interestingly, similarities were noted between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that Fanzor enzymes also employ an RNA-guided mechanism to target and cleave DNA.

In their recent study, the scientists continued investigating RNA-guided systems by isolating Fanzors from various species such as fungi, algae, amoebae, and the Northern Quahog clam. Through biochemical characterization led by co-first author Makoto Saito, the team established that Fanzor proteins are DNA-cutting endonucleases utilizing non-coding RNAs called ωRNAs to target specific sites in the genome. This discovery represents the first instance of such a mechanism observed in eukaryotes, including animals.

In contrast to CRISPR proteins, Fanzor enzymes are encoded within the eukaryotic genome as part of transposable elements. Phylogenetic analysis conducted by the team suggests that the Fanzor genes have been transferred horizontally from bacteria to eukaryotes.

Saito explained, “These OMEGA systems are more ancestral to CRISPR and they are among the most abundant proteins on the planet, so it makes sense that they have been able to hop back and forth between prokaryotes and eukaryotes.”

To evaluate the potential of Fanzor as a genome editing tool, the researchers demonstrated its ability to introduce insertions and deletions at specific genome sites in human cells. Initially, the Fanzor system exhibited lower efficiency in DNA cleavage compared to CRISPR/Cas systems. However, through systematic engineering, the team introduced specific mutations into the protein that increased its activity tenfold. Furthermore, unlike certain CRISPR systems and the OMEGA protein TnpB, the researchers observed that a fungal-derived Fanzor protein did not exhibit “collateral activity,” where the RNA-guided enzyme cleaves not only its intended DNA target but also neighboring DNA or RNA. These findings suggest the potential development of efficient genome editors using Fanzors.

Co-first author Peiyu Xu conducted an analysis of the molecular structure of the Fanzor/ωRNA complex, revealing insights into how it binds to DNA for cleavage. Fanzor exhibits structural similarities with its prokaryotic counterpart, the CRISPR-Cas12 protein, but the interaction between the ωRNA and catalytic domains of Fanzor is more extensive, suggesting a potential role for ωRNA in catalytic reactions. Xu expressed excitement about these structural insights, as they can aid in further engineering and optimization of Fanzor for improved efficiency and precision as a genome editor.

Similar to CRISPR-based systems, the Fanzor system can be easily reprogrammed to target specific genome sites. Zhang envisions that it may one day become a powerful genome editing technology for both research and therapeutic applications. The abundance of RNA-guided endonucleases like Fanzors expands our understanding of OMEGA systems across different life kingdoms and suggests the likelihood of discovering more such systems in the future.

Zhang concluded, “Nature is amazing. There’s so much diversity. There are probably more RNA-programmable systems out there, and we continue to explore and hope to discover them.” The Zhang laboratory’s ongoing research and exploration aim to uncover additional RNA-programmable systems in nature, harness their potential, and contribute to advancements in genetic medicine and genome editing technologies. The rich diversity of biological systems offers promising avenues for future discoveries and innovations in the field.

Source: Broad Institute of MIT and Harvard

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