Nature Biotechnology,
Год журнала:
2025,
Номер
unknown
Опубликована: Фев. 18, 2025
Abstract
Understanding
the
diverse
dynamic
behaviors
of
individual
RNA
molecules
in
single
cells
requires
visualizing
them
at
high
resolution
real
time.
However,
single-molecule
live-cell
imaging
unmodified
endogenous
has
not
yet
been
achieved
a
generalizable
manner.
Here,
we
present
fluorescence
situ
hybridization
(smLiveFISH),
robust
approach
that
combines
programmable
RNA-guided,
RNA-targeting
CRISPR–Csm
complex
with
multiplexed
guide
RNAs
for
direct
and
efficient
visualization
range
cell
types,
including
primary
cells.
Using
smLiveFISH,
track
native
NOTCH2
MAP1B
transcripts
living
identify
two
distinct
localization
mechanisms
cotranslational
translocation
mRNA
endoplasmic
reticulum
directional
transport
toward
periphery.
This
method
potential
to
unlock
principles
governing
spatiotemporal
organization
health
disease.
The Innovation,
Год журнала:
2024,
Номер
5(2), С. 100564 - 100564
Опубликована: Янв. 8, 2024
The
type
V-I
CRISPR-Cas
system
is
becoming
increasingly
more
attractive
for
genome
editing.
However,
natural
nucleases
of
this
often
exhibit
low
efficiency,
limiting
their
application.
Here,
we
used
structure-guided
rational
design
and
protein
engineering
to
optimize
an
uncharacterized
Cas12i
nuclease,
Cas12i3.
As
a
result,
developed
Cas-SF01,
Cas12i3
variant
that
exhibits
significantly
improved
gene
editing
activity
in
mammalian
cells.
Cas-SF01
shows
comparable
or
superior
performance
compared
SpCas9
other
Cas12
nucleases.
Compared
Cas12i3,
has
expanded
PAM
range
effectively
recognizes
NTTN
noncanonical
NATN
TTVN
PAMs.
In
addition,
identified
amino
acid
substitution,
D876R,
markedly
reduced
the
off-target
effect
while
maintaining
high
on-target
activity,
leading
development
Nature Biotechnology,
Год журнала:
2025,
Номер
unknown
Опубликована: Фев. 18, 2025
Abstract
Understanding
the
diverse
dynamic
behaviors
of
individual
RNA
molecules
in
single
cells
requires
visualizing
them
at
high
resolution
real
time.
However,
single-molecule
live-cell
imaging
unmodified
endogenous
has
not
yet
been
achieved
a
generalizable
manner.
Here,
we
present
fluorescence
situ
hybridization
(smLiveFISH),
robust
approach
that
combines
programmable
RNA-guided,
RNA-targeting
CRISPR–Csm
complex
with
multiplexed
guide
RNAs
for
direct
and
efficient
visualization
range
cell
types,
including
primary
cells.
Using
smLiveFISH,
track
native
NOTCH2
MAP1B
transcripts
living
identify
two
distinct
localization
mechanisms
cotranslational
translocation
mRNA
endoplasmic
reticulum
directional
transport
toward
periphery.
This
method
potential
to
unlock
principles
governing
spatiotemporal
organization
health
disease.
