Direct repeat region 3′ end modifications regulate Cas12a activity and expand its applications
Nucleic Acids Research,
Год журнала:
2025,
Номер
53(3)
Опубликована: Янв. 24, 2025
Abstract
CRISPR-Cas12a
technology
has
transformative
potential,
but
as
its
applications
grow,
enhancing
inherent
functionalities
is
essential
to
meet
diverse
demands.
Here,
we
reveal
a
regulatory
mechanism
for
LbCas12a
through
direct
repeat
(DR)
region
3′
end
modifications
and
de-modifications,
which
can
regulate
LbCas12a’s
cis-
trans-cleavage
activities.
We
extensively
explored
the
effects
of
introducing
phosphorylation,
DNA,
photo-cleavable
linker,
DNA
at
DR
on
functionality.
find
that
temporary
inhibitory
function
Cas12a
be
reactivated
by
modification
corresponding
substances,
such
alkaline
phosphatase
(ALP),
immunoglobulin
G
(IgG),
alpha-fetoprotein
(AFP),
exonucleases,
ultraviolet
radiation,
glycosylases,
greatly
expand
scope
application
Cas12a.
Clinical
demonstrated
promising
results
in
ALP,
AFP,
trace
Epstein–Barr
virus
detection
compared
gold
standard
methods.
Our
research
provides
valuable
insights
into
regulating
activity
significantly
expands
potential
clinical
targets,
paving
way
future
universal
clustered
regularly
interspaced
short
palindromic
repeats
(CRISPR)
diagnostic
strategies.
Язык: Английский
RNA aptamer-based CRISPR-Cas12a system for enhanced small molecule detection and point-of-care testing
International Journal of Biological Macromolecules,
Год журнала:
2025,
Номер
303, С. 140675 - 140675
Опубликована: Фев. 4, 2025
Язык: Английский
Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation
ACS Sensors,
Год журнала:
2025,
Номер
10(3), С. 2073 - 2079
Опубликована: Март 1, 2025
5-Hydroxymethylcytosine
(5hmC)
plays
a
key
role
in
the
DNA
demethylation
process
and
serves
as
stable
epigenetic
marker
human
genome
which
is
closely
associated
with
disease
progression,
particularly
diabetes,
colorectal
cancer,
liver
cancer.
However,
convenient
sensitive
methods
for
detecting
quantifying
5hmC
are
scarce,
especially
complex
biological
environments.
Herein,
novel
attempt
at
hypersensitive
quantitative
detection
of
was
presented.
A
multifunctional
photosensitive
probe
therefore
introduced
specific
labeling,
enrichment,
elution
5hmC-DNA.
Combining
isothermal
assay
leveraging
rolling
circle
amplification
Cas12a
accurate
recognition,
we
achieved
trace
amounts
level
11
fM.
Global
measured
various
samples
using
little
10
ng
input
by
real-time
PCR
instrument.
The
reported
approach
imposed
no
sequence
restrictions,
demonstrating
promising
potential
modified
bases
nucleic
acids
within
environments,
such
blood,
urine,
saliva
samples.
Язык: Английский
Expanding Cas12a Activity Control with an RNA G‐Quadruplex at the 5′ end of CRISPR RNA
Advanced Science,
Год журнала:
2024,
Номер
unknown
Опубликована: Дек. 25, 2024
Precise
control
of
Cas12a
activity
is
essential
for
the
improvement
detection
limit
clinical
diagnostics
and
minimization
errors.
This
study
addresses
challenge
controlling
activity,
especially
in
context
nucleic
acid
where
inherent
incompatibility
between
isothermal
amplification
CRISPR
reactions
complicates
accurate
diagnostics.
An
RNA
G-quadruplex
(RG4)
structure
at
5'
end
crRNA
introduced
to
modulate
accurately
without
need
chemical
modifications.
The
results
indicate
that
presence
RG4
does
not
significantly
impact
Cas12a's
cleavage
but
can
be
controlled
by
stabilizers,
enabling
suppression
subsequent
restoration
with
potential
precise
control.
Moreover,
use
expanded
incorporating
it
into
split
crRNA,
introducing
directly
direct
repeat
(DR)
region,
tailored
regulation
different
targets
matching
various
Spacer
regions.
Additionally,
a
light-controlled
one-pot
method
activating
developed,
thereby
enhancing
accuracy
sensitivity
samples.
showcases
pioneering
manipulating
streamlining
diagnostics,
paving
way
advances
testing.
Язык: Английский