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Eukaryotes Have CRISPR-Like Systems That Can Edit Genomes, MIT Teams Report


NEW YORK – Two teams of researchers at the Massachusetts Institute of Technology have demonstrated that some eukaryotic organisms have their own RNA-guided endonucleases, which likely share a common ancestor with certain Cas proteins.

"Just when you think CRISPR is plateauing, there's something new around the corner," said Omar Abudayyeh, a researcher at MIT who earlier this month posted a BioRxiv preprint on these proteins and their ubiquity in eukaryotes and their viruses.

Just like bacterial Cas9, these proteins are programmable and could be used in human genome engineering applications.

Separately, researchers led by Feng Zhang of the Broad Institute and MIT described their own analysis of these CRISPR-like systems in eukaryotes in a paper published in Nature on Wednesday. Their study characterized certain Obligate Mobile Element Guided Activity (OMEGA) systems that they first reported in 2021. This work led them to Fanzors, a type of protein associated with certain transposons, and their ability to be directed to edit human DNA.

Found in a variety of eukaryotes, from amoebae to fungi to clams, Fanzors, like Cas effectors, take looped RNAs expressed from a nearby noncoding DNA array and use it as a guide to cleave DNA at particular motifs. At roughly half the size of bacterial Cas proteins, such as Cas9, based on the number of amino acids, Fanzor systems may be more easily delivered to human cells and tissues as therapeutics.

While optimization is still needed, this new class of genome editors looks promising. "This is exciting work describing the diversity of RNA-guided DNA nucleases in eukaryotes and their potential for gene editing," said Janice Chen, chief technology officer and cofounder of Mammoth Biosciences, a CRISPR-based genome editing company, who was not involved with the studies. "There is a playbook for translating these discoveries into tools, and time will tell how Fanzors stack up against the existing gene editing toolbox."

Abudayyeh's study was done in collaboration with fellow MIT researcher Jonathan Gootenberg. Both are also former Zhang lab members. Their preprint discusses Fanzors and their common ancestor with CRISPR proteins, known as TnpBs. They proposed to name the broad class of systems HERMES, for Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems; however, they said they're not married to that term. They also showed that these protein complexes are RNA-guided, have nuclear localization sequences, and can be turned to human genome engineering. However, their systems showed very low editing efficiency.

These works build on the Zhang lab's survey of genome editing systems across the tree of life. After early work on CRISPR-Cas9 systems, especially on how they could be used to edit mammalian genomes, Zhang continued to probe for related structures. "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?'" he said in a statement. His lab led the way in developing several other classes of RNA-guided molecular systems, including those based on Cas12, Cpf1, and RNA-targeting Cas13.

The Zhang lab's discovery of OMEGAs in 2021 suggested that eukaryotic genomes, especially transposable elements, might harbor the components for RNA-guided DNA cutting enzymes.

In the new study, the researchers mined eukaryotic genomes to find Fanzors in fungi, algae, amoebae, and even a clam. Phylogenetic analysis suggests that the Fanzor genes have migrated from bacteria to eukaryotes through horizontal gene transfer.

"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," Makoto Saito, a postdoc in the Zhang lab and co-first author of the Nature paper, said in a statement.

After establishing Fanzors as RNA-guided endonucleases, the researchers showed they can generate insertions and deletions at targeted genome sites in human cells, using Fanzors from Spizellomyces punctatus, a soil fungus. Initially, they found a SpuFz enzyme to be less efficient at editing DNA than CRISPR-Cas systems, but after some optimization, they increased editing activity tenfold. Across 12 human genomic loci, an optimized Fanzor protein showed indel activity at levels around 10 to 15 percent, with one locus at nearly 20 percent.

Abudayyeh and Gootenberg, who have been independent researchers for about four years now, declined to discuss the details of their study, as it is under review. However, they spoke broadly about the topic. "Scientifically, it's a huge finding," Gootenberg said. These kinds of enzymes have been known about since 2013, but the fact that they're RNA-programmable and that they have evolved to potentially be useful as genome editors shows there is still much to be learned, he said.

Each Fanzor system appears to have its quirks, leading to differences in key characteristics, including the guide RNA sequence length and secondary structure, target-adjacent motif (TAM) recognition site, cleavage site relative to the TAM, and cleavage pattern (e.g., 5' overhang).

"We still need to engineer the enzyme further so that it will match the efficacy of the Cas9 gold standard," members of the Zhang lab team said in an email. "The compactness of Fanzor, however, makes it an attractive option for applications." They added that at least one of the Fanzor proteins does not have collateral activity, "so it might provide more targeted editing than some of the Cas12 enzymes."

Eukaryotic-derived genome editing systems could potentially be less immunogenic, Gootenberg noted.

Abudayyeh and Gootenberg have applied for a patent related to their work and are both cofounders of Sherlock Biosciences, an MIT spinout pursuing CRISPR-based diagnostics; COVID-19 testing startup Proof Diagnostics; and Tome Biosciences. However, they said they have not yet seriously considered commercialization of these new technologies.

In December, Zhang — also a cofounder of Sherlock, as well as Editas Medicine — applied for a patent on "reprogrammable Fanzor polynucleotides and uses thereof." He has also applied for a patent on "retrotransposons and use thereof." The application says that "in some embodiments, the site-specific nuclease is a 1scB or a TnpB," the likely ancestor of Fanzor proteins.

Both studies noted the abundance of RNA-guided endonucleases in all types of organisms. "Nature is amazing. There's so much diversity," Zhang said. "There are probably more RNA-programmable systems out there, and we're continuing to explore and will hopefully discover more."

"We're screening for a lot more orthologs," Abudayyeh said. Gootenberg added that the function of these enzymes in nature remains unknown. "That's a huge question, and there's a wealth of biology to find here."