How a CRISPR-Like System Was Discovered in Eukaryotes

At the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, Dr. Feng Zhang discovers and develops gene-editing systems that can be harnessed as research tools and potential therapeutics. Renowned for his innovative approaches to science and technology, Zhang is considered a leading scientist in the CRISPR research field.

After the discovery and characterization of CRISPR-Cas systems in prokaryotic organisms over a decade ago, CRISPR-based genome editing is utilized across an ever-growing list of applications, from basic research to gene therapy development and modern agriculture. Zhang has long wondered whether a similar system could exist in other kingdoms of life. Now, he has an answer.

A CRISPR-like system in eukaryotes

Last month, Zhang’s lab published what he describes as “the most comprehensive study we have reported in a single paper to date” in Nature. The paper outlines the team’s discovery and characterization of the first programmable RNA-guided system in eukaryotes, which centers around an RNA-guided endonuclease named Fanzor. Many scientists had been doubtful that such systems could exist in complex life forms. “It's typical cleverness from the Zhang lab, proving them wrong," says geneticist Ethan Bier, who was not involved in the research.

Fanzor is encoded within transposable elements of the eukaryotic genome. “We have known about eukaryotic Fanzor proteins for many years now (the protein sequences were discovered by others many years ago), but the first time we suspected that they might be RNA-guided nucleases is when we were studying the ancestry of proteins like Cas9 and Cas12 a few years ago,” says Zhang.

A likeness between eukaryotic and prokaryotic nucleases

In 2021, Zhang and colleagues were exploring the evolution and functions of IscB proteins in bacteria. IscB proteins are nucleases encoded in a family of “jumping genes” called IS200/IS605. At that point, the function and interactions of IscB had not been fully characterized. Zhang and colleagues discovered that IscB, and another IS200/IS605 transposon-encoded protein – TnpB, likely gave rise to Cas9 and Cas12. In Science, they called this class of transposon-encoded RNA-guided nucleases “OMEGA”, an acronym for obligate mobile element–guided activity.

The researchers hypothesized that TnpB may be the ancestor of the eukaryotic enzyme Fanzor, because of similarities between the two. This further drove Zhang’s conviction that eukaryotes may also have a CRISPR-Cas-like system, he describes: “Because of the conservations between TnpB and Fanzor, we had a good reason to think that Fanzor is most likely also an RNA-guided OMEGA nuclease. So, after we finished the OMEGA study on IscB and TnpB, we focused on studying Fanzor.”

Zhang and colleagues think that Fanzors could have evolved from TnpB proteins that moved from bacteria to eukaryotes in a genetic “shuffling” of sorts. Transposable elements containing TnpB may have “jumped” to these new genomes via horizontal gene transfer. “We found Fanzors in a number of eukaryotic organisms that live in symbiotic relationships with prokaryotes that contain the related TnpB proteins. We also found evidence that Fanzors spread between eukaryotes,” he says.

Fanzor is guided by RNA

The current Nature paper outlines the extensive work undertaken by the McGovern Institute team to understand Fanzor’s function, which included bioinformatic evolutionary analysis, biochemical and genetic studies, engineering and optimization of an enzyme for enhanced gene editing in human cells, and CryoEM structure determination.

The first step for the team was to address the question of whether Fanzor is actually RNA-guided. This wasn’t an easy feat, Zhang recalls, “Unlike CRISPR and OMEGA systems, the non-coding RNA for Fanzor was not easy to predict based on just looking at the genomic sequence. We had to empirically look for the guide RNA.”  

Because Fanzor is eukaryotic, it had to be studied in a eukaryotic system, adding further complexity to the work. “The model organism Saccharomyces cerevisiae (baker’s yeast) was used instead of the bacteria Escherichia coli, which is well established for testing the function of CRISPR proteins. We had to develop new assays in S. cerevisiae to study Fanzors.

A Cryo-EM map of a Fanzor protein (gray, yellow, light blue and pink) in complex with omegaRNA (purple) and its target DNA (red). Non-target DNA strand in blue. Credit: Courtesy of the Zhang lab, Broad Institute of MIT and Harvard/McGovern Institute for Brain Research at MIT.

 “We are quite excited about this paper […] Typically, a single paper would just cover one aspect of an enzyme system. For example, we have published papers that just described the biochemical characterization of a protein, or a paper that described the engineering of an enzyme for genome editing, or a paper that described the study of a protein’s structure. This Fanzor paper is like 4 papers combined into a single comprehensive paper — we have bioinformatic evolutionary analysis, biochemical and genetic studies, engineering and optimization of an enzyme for enhanced gene editing in human cells, and CryoEM structure determination. It is very satisfying to be able to report so comprehensively at once,” says Zhang.

The team discovered that Fanzor shares similarities with CRISPR in that it does interact with a guide RNA – a molecule called omegaRNA, or ωRNA. “The Fanzor protein is a kind of molecular scissors that can cut DNA. Fanzor interacts very closely with a special piece of RNA, called an omegaRNA, and this omegaRNA contains a section, called the guide. When the guideRNA sequence and the target DNA match up, they ‘zip together’ and Fanzor can then recognize and cut that specific piece of DNA,” says Zhang.

“Once we had found the non-coding RNA and had a system we could work in, from there, we were able to demonstrate the RNA-guided activity and begin engineering the system,” he adds.

A valuable new technology for human genome editing

Fanzor’s efficiency as a genome-editing tool was initially lower than that of CRISPR-Cas systems when applied in human cells. It was successful ~12% of the time, but with genome engineering enhancements, this efficiency could be increased. “We made changes to both the Fanzor protein and the omegaRNA. For the protein, we changed amino acids that we predicted were important for interacting with either the target DNA or the omegaRNA to try and increase the strength of those interaction[s],” explains Zhang. “For the omegaRNA, we tested several different variations to increase its stability in human cells. These optimizations boosted activity ~10 fold, and we are continuing to work on engineering the system for enhanced and extended function in human cells.”

Technology Networks asks Zhang the big question – does he envision that Fanzor could one day be a superior tool to CRISPR-Cas genome-editing, or will it offer a complementary approach? He emphasizes that Fanzor proteins are more compact than CRISPR proteins, which is certainly appealing from a delivery perspective; the sheer size of the CRISPR system is considered the most challenging barrier to its in vivo use.

“Further refinements to improve their targeting efficiency could make them [Fanzor proteins] a valuable new technology for human genome editing,” Zhang says. “We are excited to see how the trajectory unfolds, and we are continuing to work to develop Fanzor into a valuable new technology for human genome editing. Going forward, we are continuing to study the biology of Fanzor proteins and exploring ways that we can engineer them for use as molecular technologies.”

Professor Feng Zhang was speaking to Molly Campbell, Senior Science Writer for Technology Networks.

Reference: Saito M, Xu P, Faure G, et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. 2023. doi: 10.1038/s41586-023-06356-2