Researchers uncover a new CRISPR-like system in animals that can edit the human genome

Fanzor, the first eukaryotic RNA-guided DNA-cutting enzyme, may one day be used to edit DNA more accurately than CRISPR/Cas systems.

The McGovern Institute for Brain Research at MIT and the Broad Institute at MIT and Harvard, under the direction of Feng Zhang, have discovered the first programmable RNA-guided system in eukaryotes, which include organisms like fungi, plants, and mammals.

The team explains how the system is built on a protein known as Fanzor in a report that was just published in Nature. They demonstrated how Fanzor proteins accurately target DNA using RNA as a guide and how Fanzors may be taught to alter the human cell's genome. In comparison to CRISPR-Cas systems, the small Fanzor systems may be easier to deliver to cells and tissues as treatments. With further development to increase their targeting effectiveness, they may become an important new tool for editing the human genome.

Since prokaryotes (bacteria and other single-celled creatures without nuclei) are where CRISPR-Cas was initially identified, researchers, including those in Zhang's group, have long pondered if eukaryotes had any equivalent systems. According to the current study, RNA-guided DNA-cutting processes exist in all kingdoms of life.

"CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome," says Zhang, senior author of the study, James and Patricia Poitras Professor of Neuroscience at MIT's Department of Biological Engineering, as well as an investigator at the McGovern Institute, a core institute member at the Broad Institute, and a Howard Hughes Medical Institute investigator. Zhang is also an investigator at the McGovern Institute. "This new system is an additional method for precisely altering human cells, enhancing our current arsenal of genome editing tools."

investigating the spheres of life

The Zhang lab's main goal is to create genetic medications utilizing systems that may modify human cells by focusing on particular genes and processes. We began to wonder, "What is there beyond CRISPR, and are there other RNA-programmable systems out there in nature," according to Zhang, a number of years ago.

OMEGAs are a family of RNA-programmable prokaryotic systems that were first identified by the Zhang lab two years ago. OMEGAs are frequently associated with transposable elements, or "jumping genes," in bacterial genomes, and they are thought to be the ancestors of CRISPR-Cas systems. That research also revealed parallels between eukaryotic Fanzor proteins and prokaryotic OMEGA systems, raising the possibility that the Fanzor enzymes also employ an RNA-guided mechanism to target and cut DNA.

In the latest study, the scientists continued their work on RNA-guided systems by extracting Fanzors from clams known as northern quahogs, amoeba species, and fungus. The molecular analysis of the Fanzor proteins, undertaken by co-first author Makoto Saito of the Zhang lab, revealed that they are DNA-cutting endonuclease enzymes that utilise adjacent non-coding RNAs known as RNAs to target specific places in the genome. This process has never before been discovered in eukaryotes, which include mammals.

Since Fanzor enzymes, unlike CRISPR proteins, are encoded in transposable elements in eukaryotic genomes, the team's phylogenetic research implies that the Fanzor genes have been transferred from bacteria to eukaryotes via a process known as horizontal gene transfer.

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

With no collateral harm

The researchers showed that Fanzor can produce insertions and deletions at specific genomic regions within human cells to investigate its potential as a genome editing tool. The Fanzor system was initially discovered to be less effective than CRISPR-Cas systems at snipping DNA, but by methodical engineering, the researchers induced a combination of mutations into the protein that improved its activity 10-fold. A fungal-derived protein called Fanzor did not demonstrate "collateral activity," which is when an RNA-guided enzyme cleaves its DNA target while also destroying surrounding DNA or RNA, in contrast to several CRISPR systems and the OMEGA protein TnpB. The findings imply that efficient genome editors might be created using Fanzors.

The analysis of the Fanzor/RNA complex's molecular structure and an explanation of how it binds to DNA to cut it were led by co-first author Peiyu Xu. Although Fanzor and its prokaryotic counterpart CRISPR-Cas12 protein have structural similarities, Fanzor's contact with the RNA and catalytic domains is more extensive, suggesting that the RNA may be involved in the catalytic processes. "We are excited about these structural insights for helping us further engineer and optimize Fanzor for improved efficiency and precision as a genome editor," stated Xu.

Zhang said the Fanzor system might one day be turned into a potent new genome editing method for research and therapeutic applications since it can be readily reprogrammed to target certain genome locations, similar to CRISPR-based systems. The prevalence of RNA-guided endonucleases like Fanzors increases the number of OMEGA systems that have been identified in all kingdoms of life and raises the possibility that there are even more to be discovered.

"Nature is incredible. There is such a wide variety, adds Zhang. We are exploring and, hopefully, finding other RNA-programmable systems since there are undoubtedly more of them out there.

Guilhem Faure, Samantha Maguire, Soumya Kannan, Han Altae-Tran, Sam Vo, AnAn Desimone, and Rhiannon Macrae are some of the other writers on the study.

The Broad Institute Programmable Therapeutics Gift Donors, the Howard Hughes Medical Institute, the Poitras Center for Psychiatric Disorders Research at MIT, the K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center at MIT, The Pershing Square Foundation, William Ackman, and Neri Oxman, James and Patricia Poitras, the Asness Family Foundation, Kenneth C. Griffin, the Phillips family, David Cheng, Robert Metcalfe, and H.