bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Июнь 17, 2024
Abstract
Synonymous
mutations
are
generally
considered
neutral,
while
their
roles
in
the
human
genome
remain
largely
unexplored.
Herein,
we
employed
PEmax
system
to
create
a
library
of
297,900
epegRNAs
and
performed
extensive
screening
identify
synonymous
that
impact
cell
fitness.
While
most
found
some
can
elicit
phenotypic
effects.
By
developing
specialized
machine
learning
tool,
uncovered
on
various
biological
processes
such
as
mRNA
splicing
transcription,
supported
by
multifaceted
experimental
evidence.
Notably,
alter
RNA
folding
affect
translation,
demonstrated
PLK1_S2.
integrating
data
with
our
model,
successfully
predicted
clinically
deleterious
mutations.
This
research
deepens
understanding
provides
insights
for
clinical
disease
studies.
Cell,
Год журнала:
2024,
Номер
187(5), С. 1076 - 1100
Опубликована: Фев. 1, 2024
Genome
editing
has
been
a
transformative
force
in
the
life
sciences
and
human
medicine,
offering
unprecedented
opportunities
to
dissect
complex
biological
processes
treat
underlying
causes
of
many
genetic
diseases.
CRISPR-based
technologies,
with
their
remarkable
efficiency
easy
programmability,
stand
at
forefront
this
revolution.
In
Review,
we
discuss
current
state
CRISPR
gene
technologies
both
research
therapy,
highlighting
limitations
that
constrain
them
technological
innovations
have
developed
recent
years
address
them.
Additionally,
examine
summarize
landscape
applications
context
health
therapeutics.
Finally,
outline
potential
future
developments
could
shape
coming
years.
Nature Biotechnology,
Год журнала:
2024,
Номер
unknown
Опубликована: Март 12, 2024
Tumor
genomes
often
harbor
a
complex
spectrum
of
single
nucleotide
alterations
and
chromosomal
rearrangements
that
can
perturb
protein
function.
Prime
editing
has
been
applied
to
install
evaluate
genetic
variants,
but
previous
approaches
have
limited
by
the
variable
efficiency
prime
guide
RNAs.
Here
we
present
high-throughput
sensor
strategy
couples
RNAs
with
synthetic
versions
their
cognate
target
sites
quantitatively
assess
functional
impact
endogenous
variants.
We
screen
over
1,000
cancer-associated
variants
TP53-the
most
frequently
mutated
gene
in
cancer-to
identify
alleles
p53
function
mechanistically
diverse
ways.
find
certain
TP53
particularly
those
oligomerization
domain,
display
opposite
phenotypes
exogenous
overexpression
systems.
Our
results
emphasize
physiological
importance
dosage
shaping
native
stoichiometry
protein-protein
interactions,
establish
framework
for
studying
sequence
context
at
scale.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Ноя. 19, 2024
Perturb-seq
enabled
the
profiling
of
transcriptional
effects
genetic
perturbations
in
single
cells
but
lacks
ability
to
examine
impact
on
tissue
environments.
We
present
Perturb-DBiT
for
simultaneous
co-
sequencing
spatial
transcriptome
and
guide
RNAs
(gRNAs)
same
section
vivo
CRISPR
screen
with
genome-scale
gRNA
libraries,
offering
a
comprehensive
understanding
how
modifications
affect
cellular
behavior
architecture.
This
platform
supports
variety
delivery
vectors,
library
sizes,
preparations,
along
two
distinct
capture
methods,
making
it
adaptable
wide
range
experimental
setups.
In
applying
Perturb-DBiT,
we
conducted
un-biased
knockouts
tens
genes
or
at
genome-wide
scale
across
three
cancer
models.
mapped
all
gRNAs
individual
colonies
corresponding
transcriptomes
human
metastatic
colonization
model,
revealing
clonal
dynamics
cooperation.
also
examined
effect
perturbation
tumor
immune
microenvironment
an
immune-competent
syngeneic
uncovering
differential
synergistic
promoting
infiltration
suppression
tumors.
allows
simultaneously
evaluating
each
knockout
initiation,
development,
metastasis,
histopathology,
landscape.
Ultimately,
not
only
broadens
scope
inquiry,
lays
groundwork
developing
targeted
therapeutic
strategies.
Nature Biotechnology,
Год журнала:
2024,
Номер
unknown
Опубликована: Ноя. 12, 2024
Mutational
scanning
connects
genetic
variants
to
phenotype,
enabling
the
interrogation
of
protein
functions,
interactions
and
variant
pathogenicity.
However,
current
methodologies
cannot
efficiently
engineer
customizable
sets
diverse
in
endogenous
loci
across
cellular
contexts
high
throughput.
Here,
we
combine
cytosine
adenine
base
editors
a
prime
editor
assess
pathogenicity
broad
spectrum
epithelial
growth
factor
receptor
gene
(EGFR).
Using
pooled
editing
guide
RNA
libraries,
install
tens
thousands
spanning
full
coding
sequence
EGFR
multiple
cell
lines
role
these
tumorigenesis
resistance
tyrosine
kinase
inhibitors.
Our
scan
identifies
important
hits,
supporting
robustness
approach
revealing
underappreciated
routes
activation
drug
response.
We
anticipate
that
multimodal
precision
mutational
can
be
applied
broadly
characterize
variation
any
element
interest
at
single-nucleotide
resolution.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2023,
Номер
unknown
Опубликована: Дек. 21, 2023
Regulatory
DNA
sequences
within
enhancers
and
promoters
bind
transcription
factors
to
encode
cell
type-specific
patterns
of
gene
expression.
However,
the
regulatory
effects
programmability
such
remain
difficult
map
or
predict
because
we
have
lacked
scalable
methods
precisely
edit
quantify
in
an
endogenous
genomic
context.
Here
present
approach
measure
quantitative
hundreds
designed
sequence
variants
on
expression,
by
combining
pooled
CRISPR
prime
editing
with
RNA
fluorescence
