SAN FRANCISCO, April 19, 2019 /PRNewswire/ -- Since the
CRISPR genome editing technology was invented in 2012, it has shown
great promise to treat a number of intractable diseases. However,
scientists have struggled to identify potential off-target effects
in therapeutically relevant cell types, which remains the main
barrier to moving therapies to the clinic. Now, a group of
scientists at the Gladstone Institutes and the Innovative Genomics
Institute (IGI), with collaborators at AstraZeneca, have developed
a reliable method to do just that.
CRISPR edits a person's genome by cutting the DNA at a specific
location. The challenge is to ensure the tool doesn't also make
cuts elsewhere along the DNA—damage referred to as "off-target
effects," which could have unforeseen consequences.
In a study published in the journal Science, the two
first authors, Beeke Wienert and Stacia
Wyman, found a new way to approach the problem.
"When CRISPR makes a cut, the DNA is broken," says Wienert, PhD,
who began the work in Jacob E.
Corn's IGI laboratory and who is now a postdoctoral scholar
in Bruce R. Conklin's laboratory at
Gladstone. "So, in order to survive, the cell recruits many
different DNA repair factors to that particular site in the genome
to fix the break and join the cut ends back together. We thought
that if we could find the locations of these DNA repair factors, we
could identify the sites that have been cut by CRISPR."
To test their idea, the researchers studied a panel of different
DNA repair factors. They found that one of them, called MRE11, is
one of the first responders to the site of the cut. Using MRE11,
the scientists developed a new technique, named DISCOVER-Seq, that
can identify the exact sites in the genome where a cut has been
made by CRISPR.
"The human genome is extremely large—if you printed the entire
DNA sequence, you would end up with a novel as tall as a 16-story
building," explains Conklin, MD, senior investigator at Gladstone
and deputy director at IGI. "When we want to cut DNA with CRISPR,
it's like we're trying to remove one specific word on a particular
page in that novel."
"You can think of the DNA repair factors as different types of
bookmarks added to the book," Conklin adds. "While some may
bookmark an entire chapter, MRE11 is a bookmark that drills down to
the exact letter than has been changed."
Different methods currently exist to detect CRISPR off-target
effects. However, they come with limitations that range from
producing false-positive results to killing the cells they're
examining. In addition, the most common method used to date is
currently limited to cultured cells in the laboratory, excluding
its use in patient-derived stem cells or animal tissue.
"Because our method relies on the cell's natural repair process
to identify cuts, it has proven to be much less invasive and much
more reliable," says Corn, PhD, who now runs a laboratory at ETH
Zurich. "We were able to test our new DISCOVER-Seq method in
induced pluripotent stem cells, patient cells, and mice, and our
findings indicate that this method could potentially be used in any
system, rather than just in the lab."
The DISCOVER-Seq method, by being applied to new cell types and
systems, has also revealed new insights into the mechanisms used by
CRISPR to edit the genome, which will lead to a better
understanding of the biology of how this tool works.
"The new method greatly simplifies the process of identifying
off-target effects while also increasing the accuracy of the
results," says Conklin, who is also a professor of medical genetics
and molecular pharmacology at UC San Francisco (UCSF). "This could
allow us to better predict how genome editing would work in a
clinical setting. As a result, it represents an essential step in
improving pre-clinical studies and bringing CRISPR-based therapies
closer to the patients in need."
About the Study
The paper "Unbiased detection of CRISPR off-targets in vivo 1
using DISCOVER-Seq" was published by the journal Science on
April 19, 2019. Gladstone's
Hannah L. Watry and Luke M. Judge (who is also at UCSF) contributed
to this study. Other authors also include Christopher D. Richardson, Jonathan T. Vu, and Katelynn R. Kazane from IGI, Charles D. Yeh from ETH Zurich, as well as Pinar
Akcakaya, Michelle J. Porritt, and
Michaela Morlock from
AstraZeneca.
The work was supported by Gladstone, the National Institutes of
Health (grants EY028249 and HL13535801), the Li Ka Shing
Foundation, the Heritage Medical Research Institute, the Fanconi
Anemia Research Foundation, a Sir Keith Murdoch Fellowship from the
American Australian Association, and an Early Career Fellowship
from the National Health and Medical Research Council.
About the Gladstone Institutes
To ensure our work does the greatest good, the Gladstone
Institutes focuses on conditions with profound medical, economic,
and social impact—unsolved diseases. Gladstone is an independent,
nonprofit life science research organization that uses visionary
science and technology to overcome disease. It has an academic
affiliation with the University of California,
San Francisco.
Media Contact: Julie Langelier |
Science Writer and PR Specialist | julie.langelier@gladstone.org |
415.734.5000
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SOURCE Gladstone Institutes