Nature Biomedical Engineering,
Journal Year:
2024,
Volume and Issue:
unknown
Published: July 10, 2024
Abstract
Prime
editing
(PE)
enables
precise
and
versatile
genome
without
requiring
double-stranded
DNA
breaks.
Here
we
describe
the
systematic
optimization
of
PE
systems
to
efficiently
correct
human
cystic
fibrosis
(CF)
transmembrane
conductance
regulator
(
CFTR
)
F508del,
a
three-nucleotide
deletion
that
is
predominant
cause
CF.
By
combining
six
efficiency
optimizations
for
PE—engineered
guide
RNAs,
PEmax
architecture,
transient
expression
dominant-negative
mismatch
repair
protein,
strategic
silent
edits,
PE6
variants
proximal
‘dead’
single-guide
RNAs—we
increased
correction
efficiencies
F508del
from
less
than
0.5%
in
HEK293T
cells
58%
immortalized
bronchial
epithelial
(a
140-fold
improvement)
25%
patient-derived
airway
cells.
The
also
resulted
minimal
off-target
editing,
edit-to-indel
ratios
3.5-fold
greater
those
achieved
by
nuclease-mediated
homology-directed
repair,
functional
restoration
ion
channels
over
50%
wild-type
levels
(similar
via
combination
treatment
with
elexacaftor,
tezacaftor
ivacaftor)
primary
Our
findings
support
feasibility
durable
one-time
Science,
Journal Year:
2023,
Volume and Issue:
379(6629)
Published: Jan. 19, 2023
The
advent
of
clustered
regularly
interspaced
short
palindromic
repeat
(CRISPR)
genome
editing,
coupled
with
advances
in
computing
and
imaging
capabilities,
has
initiated
a
new
era
which
genetic
diseases
individual
disease
susceptibilities
are
both
predictable
actionable.
Likewise,
genes
responsible
for
plant
traits
can
be
identified
altered
quickly,
transforming
the
pace
agricultural
research
breeding.
In
this
Review,
we
discuss
current
state
CRISPR-mediated
manipulation
human
cells,
animals,
plants
along
relevant
successes
challenges
present
roadmap
future
technology.
Cell,
Journal Year:
2021,
Volume and Issue:
184(22), P. 5635 - 5652.e29
Published: Oct. 1, 2021
While
prime
editing
enables
precise
sequence
changes
in
DNA,
cellular
determinants
of
remain
poorly
understood.
Using
pooled
CRISPRi
screens,
we
discovered
that
DNA
mismatch
repair
(MMR)
impedes
and
promotes
undesired
indel
byproducts.
We
developed
PE4
PE5
systems
which
transient
expression
an
engineered
MMR-inhibiting
protein
enhances
the
efficiency
substitution,
small
insertion,
deletion
edits
by
average
7.7-fold
2.0-fold
compared
to
PE2
PE3
systems,
respectively,
while
improving
edit/indel
ratios
3.4-fold
MMR-proficient
cell
types.
Strategic
installation
silent
mutations
near
intended
edit
can
enhance
outcomes
evading
MMR.
Prime
editor
optimization
resulted
a
PEmax
architecture
efficacy
2.8-fold
on
HeLa
cells.
These
findings
enrich
our
understanding
establish
show
substantial
improvement
across
191
seven
mammalian
Nature Communications,
Journal Year:
2021,
Volume and Issue:
12(1)
Published: April 9, 2021
Abstract
Prime
editors
(PEs)
mediate
genome
modification
without
utilizing
double-stranded
DNA
breaks
or
exogenous
donor
as
a
template.
PEs
facilitate
nucleotide
substitutions
local
insertions
deletions
within
the
based
on
template
sequence
encoded
prime
editing
guide
RNA
(pegRNA).
However,
efficacy
of
in
adult
mice
has
not
been
established.
Here
we
report
an
NLS-optimized
SpCas9-based
editor
that
improves
efficiency
both
fluorescent
reporter
cells
and
at
endogenous
loci
cultured
cell
lines.
Using
this
system,
could
also
seed
tumor
formation
through
somatic
mouse.
Finally,
successfully
utilize
dual
adeno-associated
virus
(AAVs)
for
delivery
split-intein
demonstrate
system
enables
correction
pathogenic
mutation
mouse
liver.
Our
findings
further
establish
broad
potential
technology
directed
installation
modifications
vivo,
with
important
implications
disease
modeling
correction.
Journal of Controlled Release,
Journal Year:
2022,
Volume and Issue:
342, P. 345 - 361
Published: Jan. 10, 2022
The
discovery
of
clustered
regularly
interspaced
short
palindromic
repeats
(CRISPR)
genome
editing
technology
opened
the
door
to
provide
a
versatile
approach
for
treating
multiple
diseases.
Promising
results
have
been
shown
in
numerous
pre-clinical
studies
and
clinical
trials.
However,
safe
effective
method
deliver
genome-editing
components
is
still
key
challenge
vivo
therapy.
Adeno-associated
virus
(AAV)
one
most
commonly
used
vector
systems
date,
but
immunogenicity
against
capsid,
liver
toxicity
at
high
dose,
potential
genotoxicity
caused
by
off-target
mutagenesis
genomic
integration
remain
unsolved.
Recently
developed
transient
delivery
systems,
such
as
virus-like
particle
(VLP)
lipid
nanoparticle
(LNP),
may
solve
some
issues.
This
review
summarizes
existing
possible
solutions
overcome
their
limitations.
Also,
we
highlight
ongoing
trials
therapy
recently
tools
applications.
