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.
