Human Gene Therapy - April 2023 - 255

GENE THERAPY BRIEFS
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PASTE, Don't Cut: Genome Editing Tool Looks Beyond
CRISPR and Prime
Alex Philippidis*
Mary Ann Liebert, Inc., Publishers, New Rochelle, New York, USA.
A RECENTLY PATENTED genome editing
tool called PASTE holds genuine
promise for expanding the
universe of treatable genetic diseases.
The approach combines elements
of CRISPR and prime editing
with a pair of enzymes designed to
enable the integration of large segments
of DNA without incurring
double-stranded DNA breaks.
U.S. Patent No. 11572556,
assigned to MIT, covers systems,
methods, and compositions for programmable
addition via site-specific
targeting elements (PASTE). The
patent describes site-specific integration
of a nucleic acid into a
genome, using a CRISPR-Cas9
nickase fused to a reverse transcriptase
(RT) and a serine integrase.
These enzymes target specific
genome sequences known as attachment
sites, binding to them before
integrating their DNA payload.
PASTE can insert DNA fragments
as large as 50,000 base pairs,
which puts it on a different plane
compared with other genome editing
tools such as prime editing.
The credited inventors are Omar
Abudayyeh, PhD, and Jonathan
*Correspondence: E-mail: aphilippidis@liebertpub.com
HUMAN GENE THERAPY, VOLUME 34, NUMBERS 7 and 8
ª 2023 by Mary Ann Liebert, Inc.
DOI: 10.1089/hum.2023.29238.bfs j 255
Gootenberg, PhD, two McGovern
fellows at MIT's McGovern Institute
for Brain Research. The duo, who
were both graduate students with
CRISPR pioneer Feng Zhang, PhD,
at the Broad Institute before moving
to the McGovern Institute, detailed
PASTE in an article published past
year in Nature Biotechnology.
PASTE entails the engineering
of Cas9, RT, and integrase linkers to
create a fusion protein capable
of efficient integration (5-50%) of
diverse cargos at precisely defined
target locations within the human
genome with small stereotyped scars
that can serve as protein linkers.
The serine integrases used in
PASTE can insert DNA sequences
as large as 50,000 base pairs by targeting
specific ''attachment'' sites
within the genome.
''It's a two-step process where we
first insert the constant sequence,
which is a small insert-the site is
between 38 and 46 nucleotides
long-and it's easier to do. Then we
use that constant sequence to put in
a larger sequence. That's the concept
in a nutshell,'' Gootenberg told GEN
Edge.
Carrying out genome editing
without the need for double-stranded
breaks in DNA was first laid out by
David Liu, PhD, and colleagues at
the Broad Institute of MIT and
Harvard. Inspired by Liu's work on
base editing, Andrew Anzalone,
MD, PhD, conceived and led the
development of an RNA-based
search-and-replace genome editing
technology dubbed prime editing,
reported in a landmark 2019 article
published in Nature.
Liu cofounded Prime Medicine to
commercialize prime editing based
on Anzalone's work. In 2021, Liu's
team published the use of prime
editing to install an integrase/
recombinase landing site into a target
DNA site, followed by the catalytic
integration of cargo DNA into that
landing site. Prime Medicine has
since developed a system it calls
PASSIGE (prime-assisted sitespecific
integrase gene editing).
According to Liu, one distinction
between PASTE and PASSIGE is that
PASTE fuses the prime editor and the
integrase enzyme into a single protein
chain, whereas PASSIGE typically
uses them as two separate proteins.
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