Feng Zhang’s lab unveils new ‘Fanzor’ gene editing tool made from eukaryotic proteins

Microbes have lost their monopoly as the sole source of new enzymes that can be easily programmed to make precise edits to the genome.

Two independent teams, both led by scientists from the Massachusetts Institute of Technology, on Wednesday unveiled new gene editing tools based on proteins called ‘Fanzors’ that are found in eukaryotes, a broad branch on the tree of life that includes algae, fungi, plants, and animals.

Until now, similar DNA-altering enzymes, such as those employed in CRISPR gene editing, have only been found in single-celled microbes called prokaryotes, largely bacteria. The discovery of such enzymes in eukaryotes equips scientists with new tools that might prove to have advantages for developing therapies and suggests that more gene editing proteins may be hiding in plain sight.

“This is another example of the power of studying biodiversity,” Feng Zhang, a researcher at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT told Endpoints News in an email. “There are likely many more interesting and potentially useful systems waiting to be discovered and harnessed.”

Zhang, a pioneer of CRISPR gene editing, published a paper in Nature on Wednesday characterizing the Fanzor proteins and explaining how researchers took the first steps towards improving their natural properties. Zhang’s publication comes two weeks after his former students Omar Abudayyeh and Jonathan Gootenberg, who now share a lab at the McGovern Institute, posted their own study describing the gene editing ability of Fanzors on the preprint server bioRxiv.

“People have always wondered if there are eukaryotic versions of CRISPR,” Abudayyeh said in an interview. “This is checking a box that people thought might exist, but no one knew how. It is kind of crazy that these are cousins of CRISPR.”

Omar Abudayyeh

The discoveries are the latest in a gold rush to discover new CRISPR enzymes or other proteins that could be turned into new-and-improved gene editors. Scientists still don’t know what purpose these Fanzor proteins serve in nature, but the new technology is sure to attract the attention of private investors, and Abudayyeh, Gootenberg, and Zhang have collectively launched at least eight biotech companies based on their gene editing tools or similar technologies. Although all three researchers said it was “too early” to form a new company around Fanzors.

“Although more research is needed to show whether this new system is better than the widely used CRISPR/Cas9 for human gene editing, it holds a lot of promise for future clinical applications,” Imran Noorani, a researcher at the University College London, said in an email.

‘Jumping genes’

The two groups found that Fanzors are DNA-cutting enzymes, or nucleases, guided by an RNA molecule that researchers can easily program to make changes to particular lines of code in the genome. If that sounds familiar, it’s because the same description applies the Cas enzymes used in CRISPR gene editing.

Fanzors were discovered and named by other researchers a decade ago but have remained overlooked until there popped up in a 2021 study from Zhang’s group. That year, his team reported the discovery of a new class of RNA-guided nucleases — broadly dubbed OMEGA — in parts of microbial genomes known as transposons, also called ‘jumping genes’ because they can move and copy and paste themselves throughout the genome.

Molecular genealogy studies revealed that one of those OMEGA proteins, called TnpB, was likely the ancestor of CRISPR-Cas12 in microbes, and possibly the ancestor of the eukaryotic protein Fanzor as well. That finding, which suggested that Fanzor was a distant cousin of CRISPR, led Zhang to wonder if it had gene-editing capabilities too.

Trawling through publicly available genetics databases, Zhang’s team found evidence of Fanzor proteins in an array of organisms including algae, fungi, plants, and certain mollusks, including the Northern quahog clam. They also found the proteins in some viruses, including the so-called giant viruses.

Molecular studies of the proteins found grooves resembling the ones that that guide RNAs slide into to direct the editing of Cas enzymes. That led the researchers to look for similar RNA molecules that could direct Fanzor to cut DNA.

Ailong Ke

Those molecules were discovered bound to Fanzor. And in 3 of 4 proteins tested, the RNA-enzyme pair could edit specific lines of DNA code with up to 11.8% efficiency, depending on the protein used and the gene it was targeting. That’s a level that’s comparable to early versions of CRISPR gene editing, but lower than state-of-the-art editors.

Ailong Ke, a researcher at Cornell University said in an email that the study was “exciting” and “exceptionally elegant.”

“RNA-guided nucleases are more abundant and more widespread than we anticipated,” he said. “They are not unique to CRISPR systems, nor prokaryotic transposons. There are likely more out there.”

Engineering a better editor

Abudayyeh and Gootenberg’s study largely focused on searching for and classifying different types of Fanzor proteins in eukaryotes, which they renamed HERMES, for Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems.

Jonathan Gootenberg

They also showed that the proteins could be used for gene editing, although with lower efficiency than in Zhang’s study. Abudayyeh and Gootenberg said they could not discuss the details of their study because it was under review for publication at a scientific journal.

Zhang’s team took the work a step further by engineering a Fanzor protein to make a better gene editor, initially focused on an enzyme from the soil fungus Spizellomyces punctatus. They landed on a trio of three mutations that improved the protein, allowing it to achieve 18.4% editing efficiency for one target, although it still only made an edit roughly 10 to 15% of the time for most targets.

“We still need to engineer the enzyme further so that it will match the efficacy of the Cas9 gold standard,” Zhang said. “One unique aspect we found with one of the Fanzor proteins (not all of them), is that unlike Cas12 proteins, the SpuFanzor does not have collateral activity. So it might provide more targeted editing than some of the Cas12 enzymes.”

The small size of Fanzor proteins, ranging from 400 to 700 amino acids, makes them attractive, Zhang said. The Cas9 proteins most commonly employed in CRISPR can range from roughly 1,000 to 1,600 amino acids, and the larger the editor, the harder it can be to deliver into the body.

The size limitation could be particularly important for next-generation versions of CRISPR such as base editing and prime editing, which combine a nuclease like Cas9 with other enzymes for more fine-tuned editing.

Ke noted that the gene editing systems derived from eukaryotes have presumably evolved to work well in the large and complex genomes. “They could be more efficient or even more precise for eukaryotic gene editing.”

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