Foodborne
pathogens,
including
Vibrio
spp.
and
norovirus,
cause
substantial
economic
healthcare
burdens
worldwide.
Rapid
sensitive
point-of-care
testing
on-farm
or
restaurants
for
batch
inspection
of
pathogenic
contamination
in
raw
food
products
is
essential.
Here,
we
present
an
easy-to-design,
cost-effective
PAM-less
Exonuclease-assisted
Cas12A
Nucleic-acid
Detection
(PECAN)
assay
paired
with
nucleic
acid
amplification
systems
rapid
visual
detection
2
species:
parahaemolyticus
(TDH)
Cholerae
(ctxA)
without
protospacer
adjacent
motif
(PAM)
site
limitation.
With
T7
exonuclease,
could
be
achieved
a
low
concentration
cas12a,
costing
$0.8
USD
per
reaction.
The
system
also
adapted
cas12a
in-field
in-lab
DNA
RNA
detection.
Angewandte Chemie,
Год журнала:
2024,
Номер
136(20)
Опубликована: Март 22, 2024
Abstract
The
CRISPR‐Cas12a
system
has
emerged
as
a
powerful
tool
for
next‐generation
nucleic
acid‐based
molecular
diagnostics.
However,
it
long
been
believed
to
be
effective
only
on
DNA
targets.
Here,
we
investigate
the
intrinsic
RNA‐enabled
trans‐
cleavage
activity
of
AsCas12a
and
LbCas12a
discover
that
they
can
directly
activated
by
full‐size
RNA
targets,
although
exhibits
weaker
than
both
single‐stranded
substrates.
Remarkably,
find
RNA‐activated
Cas12a
possesses
higher
specificity
in
recognizing
mutated
target
sequences
compared
activation.
Based
these
findings,
develop
“
U
niversal
N
uclease
I
dentification
V
irus
E
mpowered
R
NA‐
Se
nsing”
(UNIVERSE)
assay
acid
testing.
We
incorporate
T7
transcription
step
into
this
assay,
thereby
eliminating
requirement
protospacer
adjacent
motif
(PAM)
sequence
target.
Additionally,
successfully
detect
multiple
PAM‐less
targets
HIV
clinical
samples
are
undetectable
conventional
based
double‐stranded
activation,
demonstrating
unrestricted
selection
with
UNIVERSE
assay.
further
validate
utility
testing
HPV
16
samples.
envision
targeting
capability
may
bring
paradigm
shift
Cas12a‐based
detection
enhance
understanding
CRISPR‐Cas
biochemistry.
Foodborne
pathogens,
including
Vibrio
spp.
and
norovirus,
cause
substantial
economic
healthcare
burdens
worldwide.
Rapid
sensitive
point-of-care
testing
on-farm
or
restaurants
for
batch
inspection
of
pathogenic
contamination
in
raw
food
products
is
essential.
Here,
we
present
an
easy-to-design,
cost-effective
PAM-less
Exonuclease-assisted
Cas12A
Nucleic-acid
Detection
(PECAN)
assay
paired
with
nucleic
acid
amplification
systems
rapid
visual
detection
2
species:
parahaemolyticus
(TDH)
Cholerae
(ctxA)
without
protospacer
adjacent
motif
(PAM)
site
limitation.
With
T7
exonuclease,
could
be
achieved
a
low
concentration
cas12a,
costing
$0.8
USD
per
reaction.
The
system
also
adapted
cas12a
in-field
in-lab
DNA
RNA
detection.
