CRISPR/Pepper‐tDeg: A Live Imaging System Enables Non‐Repetitive Genomic Locus Analysis with One Single‐Guide RNA
Meng Chen,
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Xing Huang,
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Yakun Shi
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et al.
Advanced Science,
Journal Year:
2024,
Volume and Issue:
11(32)
Published: June 26, 2024
Abstract
CRISPR‐based
genomic‐imaging
systems
have
been
utilized
for
spatiotemporal
imaging
of
the
repetitive
genomic
loci
in
living
cells,
but
they
are
still
challenged
by
limited
signal‐to‐noise
ratio
(SNR)
at
a
non‐repetitive
locus.
Here,
an
efficient
system
is
proposed,
termed
CRISPR/Pepper‐tDeg,
engineering
CRISPR
sgRNA
scaffolds
with
degron‐binding
Pepper
aptamers
binding
fluorogenic
proteins
fused
Tat
peptide
derived
degron
domain
(tDeg).
The
target‐dependent
stability
switches
both
and
protein
allow
this
to
image
telomeres
sensitively
5‐fold
higher
SNR
than
conventional
CRISPR/MS2‐MCP
using
“always‐on”
fluorescent
tag.
Subsequently,
CRISPR/Pepper‐tDeg
applied
simultaneously
label
track
two
different
loci,
centromeres,
cells
combining
systems.
Given
further
improved
split
design,
extended
sequence
only
one
aptamer
insertions.
Neither
complex
design
nor
difficult
plasmid
construction
required,
greatly
reducing
technical
barriers
define
organization
dynamics
thus
demonstrating
large
application
potential
biological
research,
clinical
diagnosis
therapy.
Language: Английский
Engineered CRISPR/Cas Ribonucleoproteins for Enhanced Biosensing and Bioimaging
Linxin Cao,
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Zeyuan Wang,
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Chunyang Lei
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et al.
Analytical Chemistry,
Journal Year:
2025,
Volume and Issue:
unknown
Published: March 11, 2025
CRISPR-Cas
systems
represent
a
highly
programmable
and
precise
nucleic
acid-targeting
platform,
which
has
been
strategically
engineered
as
versatile
toolkit
for
biosensing
bioimaging
applications.
Nevertheless,
their
analytical
performance
is
constrained
by
inherent
functional
activity
limitations
of
natural
CRISPR/Cas
systems,
underscoring
the
critical
role
molecular
engineering
in
enhancing
capabilities.
This
review
comprehensively
examines
recent
advancements
ribonucleoproteins
(RNPs)
to
enhance
capabilities
advanced
detection
cellular
imaging.
We
explore
innovative
strategies
developing
enhanced
RNPs,
including
Cas
protein
through
mutagenesis
fusion
techniques,
guide
RNA
via
chemical
structural
modifications.
Furthermore,
we
evaluate
these
RNPs'
applications
sensitive
biomarker
live-cell
genomic
DNA
monitoring,
while
analyzing
current
challenges
prospective
developments
RNP
bioimaging.
Language: Английский
Establishment of CRISPR‐STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica
Yaru Chen,
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Mengxu Li,
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Xun Liu
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et al.
Microbial Biotechnology,
Journal Year:
2025,
Volume and Issue:
18(4)
Published: April 1, 2025
ABSTRACT
The
ability
to
regulate
gene
expression
in
multiple
directions
is
crucial
maximise
the
production
of
microbial
cell
factories.
However,
lack
a
regulatory
tool
that
can
simultaneously
activate
and
repress
genes
restricts
manipulation
diversity
Yarrowia
lipolytica
,
which
an
industrial
workhorse
for
bioproduction.
To
address
this
issue,
we
developed
CRISPR
s
caffold
RNAs
(scRNAs)‐mediated
t
ranscriptional
ctivation
r
epression
(CRISPR‐STAR)
platform.
Firstly,
evaluated
different
methods
bidirectional
regulation
using
on
both
endogenous
synthetic
promoters
Y.
chose
utilisation
orthogonal
scRNAs
recruit
activation
inhibition
domains.
Secondly,
CRISPR‐STAR
was
optimised
by
introduction
alternative
dCas
proteins,
scRNA
structures
activators.
2.6‐fold
54.9‐fold
were
achieved
promoters,
respectively,
when
VPR
transcriptional
activator
recruited
via
MS2
hairpin.
repression
several
successfully
achieved,
with
levels
ranging
from
3%
32%,
MXI1
repressor
PP7
Finally,
applied
enhance
fatty
alcohol
activating
FAR
(encodes
acyl‐CoA
reductase)
PEX10
integral
membrane
protein
required
peroxisome
biogenesis
matrix
import).
Compared
non‐targeting
control,
bidirectionally
regulated
strain
showed
55.7%
increase
yield
778.8
mg/L.
Our
findings
demonstrate
platform
enables
multi‐mode
genes,
offering
engineering
opportunities
improve
productive
performance
Y
.
Language: Английский
Aptamer–ODN Chimeras: Enabling Cell-Specific ODN Targeting Therapy
Xia Bei,
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Qubo Zhu
No information about this author
Cells,
Journal Year:
2025,
Volume and Issue:
14(10), P. 697 - 697
Published: May 12, 2025
Oligonucleotides
(ODNs)
such
as
siRNA,
saRNA,
and
miRNA
regulate
gene
expression
through
a
variety
of
molecular
mechanisms
show
unique
potential
in
the
treatment
genetic
diseases
rare
diseases,
but
their
clinical
application
is
still
limited
by
efficiency
delivery
system,
especially
problem
insufficient
targeting
extrahepatic
tissues.
As
homologous
nucleic
acid
molecules,
aptamers
have
become
key
tool
to
improve
targeted
ODNs.
Aptamer-ODN
chimeras
can
not
only
bind
multiple
proteins
on
cell
surface
with
high
specificity
selectivity,
they
also
internalize
into
cells.
Furthermore,
outperform
traditional
systems
terms
cost-effectiveness
chemical
modification
flexibility.
This
review
systematically
summarizes
origin
progress
aptamer-ODN
chimera
therapy,
discusses
some
innovative
design
strategies,
proposes
views
future
direction
chimeras.
Language: Английский