A team of researchers led by Feng Zhang at the McGovern Institute for Brain Research at MIT and the Broad Institute of MIT and Harvard has uncovered the first RNA-guided system in eukaryotes – organisms that include fungi, plants, and animals.
In a study published today in Nature, the team explains how this system is based on a protein called Fanzor. They showed that Fanzor proteins use RNA as a guide to precisely target DNA, and that Fanzors can be reprogrammed to alter the genetic makeup of human cells. Fanzor fusion systems can be delivered more easily into cells and tissues as therapeutic options than CRISPR-Cas systems, and further refinements to improve their control could make them an important new technology for editing the human genome.
CRISPR-Cas was first discovered in prokaryotes (bacteria and other single-celled organisms that lack nuclei) and scientists including those in Zhang’s lab have long wondered whether similar systems exist in eukaryotes. New research shows that RNA-guided DNA cutting mechanisms are present in all living kingdoms.
“CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different locations in the genome,” says Zhang, lead author of the study, the James and Patricia Poitras Professor of Neuroscience in MIT’s departments of Biological Engineering and Brain and Cognitive Sciences, a researcher at MIT’s McGovern Institute, a senior member of the Broad Institute, and a researcher at the Howard Hughes Medical Institute. “This new system is another way to make real changes in human cells, complementing the genome editing tools we already have.”
Exploring the realms of life
Zhang’s lab’s main goal is to develop genetic medicine using machines that can modify human cells by targeting genes and other processes. Zhang said: “A few years ago, we started asking, ‘What else is there to do with CRISPR?’
Two years ago, members of Zhang’s laboratory discovered a group of RNA-editing machines in prokaryotes called OMEGAs, which are often associated with changeable elements, or “jump genes,” in bacterial populations and may have led to CRISPR- Cas. This work also highlighted similarities between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that Fanzor enzymes may also use an RNA-guided pathway to target and cut DNA.
In the new study, the researchers continued their work on RNA-guided systems by isolating Fanzors from fungi, algae, and amoeba species, including a clam known as the Northern quahog. Co-first author Makoto Saito of Zhang’s lab led the biochemical characterization of Fanzor proteins, showing that they are DNA-cutting endonuclease enzymes that use nearby non-coding RNAs known as ωRNAs to target specific sites in the genome. It is the first time that this mechanism has been found in eukaryotes, such as animals.
Unlike CRISPR proteins, Fanzor enzymes are stored in the eukaryotic genome within transposable elements, and the team’s research shows that Fanzor genes moved from bacteria to eukaryotes through so-called horizontal gene transfer.
“These OMEGA machines are the ancestors of CRISPR and are among the most abundant proteins in the world, so it makes sense that they were able to jump between prokaryotes and eukaryotes,” says Saito.
No collateral damage
To explore Fanzor’s potential as a genome editing tool, the researchers demonstrated that it can create insertions and deletions at sites that target the genome within human cells. The researchers found that the Fanzor system was initially less efficient at capturing DNA than the CRISPR-Cas system, but through systematic engineering, they introduced a combination of protein mutations that increased its efficiency 10 times. In addition, unlike other CRISPR systems, the OMEGA protein TnpB, the team found that the Fanzor protein from fungi did not show “rental activity,” where the RNA-guided enzyme cuts the target DNA and destroys nearby DNA or RNA. The results suggest that Fanzors can be engineered as efficient genome editors.
First co-author Peiyu Xu led the experiment to analyze the molecular structure of the Fanzor/ωRNA complex and show how it interacts with DNA to cut it. Fanzor shares similarities with its prokaryotic counterpart the CRISPR-Cas12 protein, but the interaction between ωRNA and Fanzor’s promoter regions is greater, suggesting that ωRNA may play a role in the promoter. “We are excited about the information to help further engineer and improve Fanzor’s efficiency and accuracy as a genome editor,” Xu said.
Like the CRISPR system, Fanzor’s system can be easily reprogrammed to fit specific areas of the genome, and Zhang said it could one day be developed into a new genome-editing technology for research and clinical applications. The abundance of RNA-guided endonucleases such as Fanzors further expands the number of OMEGA systems known in all living kingdoms and suggests that there are many more still to be discovered.
“Nature is amazing. There are many types,” says Zhang. “There are probably many RNA editing machines out there, and we are continuing to research and we hope to find more.”
Other authors of the paper are Guilhem Faure, Samantha Maguire, Soumya Kannan, Han Altae-Tran, Sam Vo, AnAn Desimone, and Rhiannon Macrae.
Support for this work was provided by the Howard Hughes Medical Institute; Poitras Center for Psychiatric Disorders Research at MIT; K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center at MIT; Broad Institute Programmable Therapeutics Grants; Pershing Square Foundation, William Ackman, and Neri Oxman; James and Patricia Poitras; BT Charitable Foundation; Asness Family Foundation; Kenneth C. Griffin; the Phillips family; David Cheng; Robert Metcalfe; and Hugo Shong.
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