Study of DNA repair boosts prospects for gene editing technology
New tool offers ways to improve CRISPR gene-editing method

Date:
October 20, 2021
Source:
Princeton University
Summary:
Researchers have developed a new method to profile the activity
of cellular genes involved in correcting DNA damage, and applied
this method to pave the way for dramatic improvements to genome
editing technologies.



FULL STORY ==========================================================================
The ability to edit the genome by altering the DNA sequence inside a
living cell is powerful for research and holds enormous promise for the treatment of diseases. However, existing genome editing technologies
frequently result in unwanted mutations or can fail to introduce any
changes at all. These problems have kept the field from reaching its
full potential.


==========================================================================
Now, new research from the laboratory of Princeton University researcher
Britt Adamson, conducted with collaborators in the lab of Jonathan
Weissman, a member of Whitehead Institute and a professor of biology at
the Massachussetts Insittute of Technology and an investigator with the
Howard Hughes Medical Institute, and Cecilia Cotta-Ramusino, formerly at
Editas Medicine, details a novel method called Repair-seq that reveals
in exquisite detail how genome editing tools work.

"We've known for a long time that the mechanisms involved in fixing broken
DNA are essential for genome editing because to change the sequence of
DNA you first have to break it," said Britt Adamson, senior author on the
study and assistant professor in the Princeton Department of Molecular
Biology and the Lewis-Sigler Institute of Integrative Genomics. "But those processes are incredibly complex and thus often difficult to untangle."
To repair DNA, cells use many different mechanisms, each involving sets of genes working together in distinct pathways. Repair-seq allows researchers
to probe the contribution of these pathways to repair of specific DNA
lesions by simultaneously profiling how hundreds of individual genes
affect mutations produced at damaged sites. The researchers can then
generate mechanistic models of DNA repair and learn how those mechanisms
impact genome editing. Adamson and colleagues applied their method to
one of the most commonly used genome editing approaches, CRISPR-Cas9,
which employs the bacterial Cas9 nuclease to cut across both strands of
the double-stranded DNA molecule, creating lesions called double-strand
breaks.

"Editing with double-strand breaks has been the bread and butter of genome editing for a long time, but making intended changes without unwanted
mutations has been an enormous challenge," said the study's first author Jeffrey Hussmann, who conducted the work while a postdoctoral researcher
in the laboratory of Jonathan Weissman. "We set out to understand the mechanisms behind as many of the induced mutations as possible, reasoning
that this could help us optimize the system." Repair-seq experiments
generate an enormous amount of data. Analysis of that data, led by
Hussmann, produced a map of how different DNA repair pathways are linked
to particular types of Cas9-induced mutations. Building on a rich history
of research in the field, Hussmann's analysis illuminated pathways that
were already known, and identified new ones, which together highlight
the enormous complexity and myriad of systems involved in double-strand
break repair. The deep set of data unearthed in this work is now posted
on an online portal that others can use to interrogate DNA repair genes
and pathways.



========================================================================== Separately, a team led by David Liu at the Broad Institute of MIT and
Harvard developed a genome editing system called "prime editing" that
doesn't rely on creating double-strand breaks. Prime editing efficiencies
vary widely by cell type and target site, but the researchers suspected
that identifying the DNA repair pathways involved might help identify
avenues for improvement. With this in mind, Adamson and Hussmann
joined forces with Liu and colleagues to investigate prime editing
using Repair-seq.

"Working together was a huge benefit," said Adamson. "For us, it was
a fantastic experience of collaborative and team-oriented science."
The collaborating researchers found that the ability to obtain intended
edits with prime editing was affected by proteins in the DNA mismatch
repair pathway.

They then showed that inhibiting or evading that pathway dramatically
enhanced the efficiency and accuracy of prime editing outcomes --
positioning prime editing to become a more broadly applicable genome
editing technology.

"Working with Britt, Jonathan, and their labs has been a beautiful
integration of basic science, tool application, and technology development
-- a real testament to the power of multidisciplinary collaboration,"
Liu said.

Importantly, this work also demonstrates how Repair-seq can be used to
improve other genome-editing technologies. In fact, the collaborating researchers have already applied it to a third genome editing system,
which was also developed by scientists working under Liu. Results from
that study were recently published in the journal Nature Biotechnology.



========================================================================== "Repair-seq is a beautiful marriage of technological savvy and biological insight," saidJohn Doench, director of research and development in the
Genetic Perturbation Program at the Broad Institute, who was not involved
with the work.

"And for the work on prime editing, what a wonderful example of
collaboration! Prime editors have often proven difficult to work with,
and this paper starts to understand why, while also kickstarting novel solutions," he added.

Moving forward, the team will continue to improve the platform and apply
it to additional genome editing technologies.

"We see Repair-seq as a tool that allows you to take a detailed picture
of what genome editors are doing inside cells and then very quickly
assess, 'Is this a landscape in which I can find design principles that
will help improve the tool?'" Adamson said. "We are really excited to
explore future applications." The studies were supported by grants from
the National Institutes of Health, the Howard Hughes Medical Institute,
the Searle Scholars Program, the National Science Foundation, the Damon
Runyon Cancer Research Foundation, the China Scholarship Council, and
the National Cancer Institute.

========================================================================== Story Source: Materials provided by Princeton_University. Note: Content
may be edited for style and length.


========================================================================== Journal References:
1. Jeffrey A. Hussmann, Jia Ling, Purnima Ravisankar, Jun Yan, Ann
Cirincione, Albert Xu, Danny Simpson, Dian Yang, Anne Bothmer,
Cecilia Cotta-Ramusino, Jonathan S. Weissman, Britt Adamson. Mapping
the genetic landscape of DNA double-strand break repair. Cell,
2021 DOI: 10.1016/ j.cell.2021.10.002
2. Peter J. Chen, Jeffrey A. Hussmann, Jun Yan, Friederike Knipping,
Purnima
Ravisankar, Pin-Fang Chen, Cidi Chen, James W. Nelson, Gregory
A. Newby, Mustafa Sahin, Mark J. Osborn, Jonathan S. Weissman,
Britt Adamson, David R. Liu. Enhanced prime editing systems by
manipulating cellular determinants of editing outcomes. Cell,
2021; DOI: 10.1016/ j.cell.2021.09.018 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/10/211020135926.htm

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