Nature Structural & Molecular Biology,
Год журнала:
2023,
Номер
30(8), С. 1172 - 1182
Опубликована: Июль 17, 2023
Abstract
RNA-guided
type
V
CRISPR–Cas12
effectors
provide
adaptive
immunity
against
mobile
genetic
elements
(MGEs)
in
bacteria
and
archaea.
Among
diverse
Cas12
enzymes,
the
recently
identified
Cas12m2
(CRISPR–Cas
V-M)
is
highly
compact
has
a
unique
RuvC
active
site.
Although
non-canonical
triad
does
not
permit
dsDNA
cleavage,
still
protects
invading
MGEs
through
transcriptional
silencing
by
strong
DNA
binding.
However,
molecular
mechanism
of
genome
inactivation
remains
unknown.
Here
we
report
cryo-electron
microscopy
structures
two
states
Cas12m2–CRISPR
RNA
(crRNA)–target
ternary
complexes
Cas12m2–crRNA
binary
complex,
revealing
structural
dynamics
during
crRNA–target
heteroduplex
formation.
The
indicate
that
non-target
strand
tightly
bound
to
arginine-rich
cluster
recognition
(REC)
domains
site
domain,
ensuring
DNA-binding
affinity
Cas12m2.
Furthermore,
comparison
with
TnpB,
putative
ancestor
suggests
interaction
characteristic
coiled-coil
REC2
insertion
protospacer-adjacent
motif-distal
region
crucial
for
engage
immunity.
Collectively,
our
findings
improve
mechanistic
understanding
CRISPR–Cas
insights
into
evolution
TnpB
enzymes.
Nature,
Год журнала:
2022,
Номер
603(7900), С. 343 - 347
Опубликована: Март 2, 2022
Abstract
CRISPR–Cas9
as
a
programmable
genome
editing
tool
is
hindered
by
off-target
DNA
cleavage
1–4
,
and
the
underlying
mechanisms
which
Cas9
recognizes
mismatches
are
poorly
understood
5–7
.
Although
variants
with
greater
discrimination
against
have
been
designed
8–10
these
suffer
from
substantially
reduced
rates
of
on-target
5,11
Here
we
used
kinetics-guided
cryo-electron
microscopy
to
determine
structure
at
different
stages
mismatch
cleavage.
We
observed
distinct,
linear
conformation
guide
RNA–DNA
duplex
formed
in
presence
mismatches,
prevents
activation.
canonical
kinked
facilitates
cleavage,
observe
that
substrates
contain
distal
protospacer
adjacent
motif
stabilized
reorganization
loop
RuvC
domain.
Mutagenesis
mismatch-stabilizing
residues
reduces
but
maintains
rapid
By
targeting
regions
exclusively
involved
tolerance,
provide
proof
concept
for
design
next-generation
high-fidelity
variants.
Cell,
Год журнала:
2022,
Номер
185(24), С. 4574 - 4586.e16
Опубликована: Ноя. 1, 2022
CRISPR-Cas
systems
are
host-encoded
pathways
that
protect
microbes
from
viral
infection
using
an
adaptive
RNA-guided
mechanism.
Using
genome-resolved
metagenomics,
we
find
CRISPR
also
encoded
in
diverse
bacteriophages,
where
they
occur
as
divergent
and
hypercompact
anti-viral
systems.
Bacteriophage-encoded
belong
to
all
six
known
types,
though
some
lack
crucial
components,
suggesting
alternate
functional
roles
or
host
complementation.
We
describe
multiple
new
Cas9-like
proteins
44
families
related
type
V
systems,
including
the
Casλ
nuclease
family.
Among
most
of
enzymes
identified,
recognizes
double-stranded
DNA
a
uniquely
structured
RNA
(crRNA).
The
Casλ-RNA-DNA
structure
determined
by
cryoelectron
microscopy
reveals
compact
bilobed
architecture
capable
inducing
genome
editing
mammalian,
Arabidopsis,
hexaploid
wheat
cells.
These
findings
reveal
source
phages
highlight
their
value
editors
plant
human
Military Medical Research,
Год журнала:
2023,
Номер
10(1)
Опубликована: Июль 17, 2023
Clustered
regulatory
interspaced
short
palindromic
repeats
(CRISPR)
has
changed
biomedical
research
and
provided
entirely
new
models
to
analyze
every
aspect
of
sciences
during
the
last
decade.
In
study
cancer,
CRISPR/CRISPR-associated
protein
(Cas)
system
opens
avenues
into
issues
that
were
once
unknown
in
our
knowledge
noncoding
genome,
tumor
heterogeneity,
precision
medicines.
CRISPR/Cas-based
gene-editing
technology
now
allows
for
precise
permanent
targeting
mutations
provides
an
opportunity
target
small
non-coding
RNAs
such
as
microRNAs
(miRNAs).
However,
development
effective
safe
cancer
gene
editing
therapy
is
highly
dependent
on
proper
design
be
innocuous
normal
cells
prevent
introducing
other
abnormalities.
This
aims
highlight
cutting-edge
approaches
cancer-gene
based
CRISPR/Cas
miRNAs
therapy.
Furthermore,
we
potential
challenges
CRISPR/Cas-mediated
miRNA
offer
advanced
strategies
overcome
them.
Biochemistry,
Год журнала:
2023,
Номер
62(24), С. 3465 - 3487
Опубликована: Май 16, 2023
CRISPR
systems
mediate
adaptive
immunity
in
bacteria
and
archaea
through
diverse
effector
mechanisms
have
been
repurposed
for
versatile
applications
therapeutics
diagnostics
thanks
to
their
facile
reprogramming
with
RNA
guides.
RNA-guided
CRISPR-Cas
targeting
interference
are
mediated
by
effectors
that
either
components
of
multisubunit
complexes
class
1
or
multidomain
single-effector
proteins
2.
The
compact
2
broadly
adopted
multiple
applications,
especially
genome
editing,
leading
a
transformation
the
molecular
biology
biotechnology
toolkit.
diversity
enzymes,
initially
limited
Cas9
nuclease,
was
substantially
expanded
via
computational
metagenome
mining
include
numerous
variants
Cas12
Cas13,
providing
substrates
development
versatile,
orthogonal
tools.
Characterization
these
uncovered
many
new
features,
including
distinct
protospacer
adjacent
motifs
(PAMs)
expand
space,
improved
editing
specificity,
rather
than
DNA
targeting,
smaller
crRNAs,
staggered
blunt
end
cuts,
miniature
promiscuous
cleavage,
etc.
These
unique
properties
enabled
such
as
harnessing
RNase
activity
type
VI
effector,
supersensitive
nucleic
acid
detection.
well,
despite
challenge
expressing
delivering
multiprotein
effectors.
rich
enzymes
led
rapid
maturation
toolbox,
capabilities
gene
knockout,
base
prime
insertion,
imaging,
epigenetic
modulation,
transcriptional
editing.
Combined
rational
design
engineering
associated
RNAs,
natural
related
bacterial
provides
vast
resource
expanding
repertoire
tools
biotechnology.
Nature,
Год журнала:
2023,
Номер
613(7944), С. 582 - 587
Опубликована: Янв. 4, 2023
Abstract
Cas12a2
is
a
CRISPR-associated
nuclease
that
performs
RNA-guided,
sequence-nonspecific
degradation
of
single-stranded
RNA,
DNA
and
double-stranded
following
recognition
complementary
RNA
target,
culminating
in
abortive
infection
1
.
Here
we
report
structures
binary,
ternary
quaternary
complexes
to
reveal
complete
activation
pathway.
Our
autoinhibited
until
binding
cognate
which
exposes
the
RuvC
active
site
within
large,
positively
charged
cleft.
Double-stranded
substrates
are
captured
through
duplex
distortion
local
melting,
stabilized
by
pairs
‘aromatic
clamp’
residues
crucial
for
vivo
immune
system
function.
work
provides
structural
basis
this
mechanism
achieve
population-level
immunity,
can
be
leveraged
create
rational
mutants
degrade
spectrum
collateral
substrates.
Signal Transduction and Targeted Therapy,
Год журнала:
2024,
Номер
9(1)
Опубликована: Фев. 26, 2024
Precise
genome-editing
platforms
are
versatile
tools
for
generating
specific,
site-directed
DNA
insertions,
deletions,
and
substitutions.
The
continuous
enhancement
of
these
has
led
to
a
revolution
in
the
life
sciences,
which
promises
deliver
novel
therapies
genetic
disease.
can
be
traced
back
1950s
with
discovery
DNA's
double-helix
and,
after
70
years
development,
evolved
from
crude
vitro
applications
wide
range
sophisticated
capabilities,
including
vivo
applications.
Nonetheless,
precise
faces
constraints
such
as
modest
efficiency,
delivery
challenges,
off-target
effects.
In
this
review,
we
explore
genome-editing,
focus
on
introduction
landmark
events
its
history,
various
platforms,
systems,
First,
discuss
history
genome-editing.
Second,
describe
current
state
strategies
explain
how
techniques
offer
unprecedented
precision
versatility
modifying
human
genome.
Third,
introduce
systems
used
deploy
components
through
DNA,
RNA,
RNPs.
Finally,
summarize
labeling
endogenous
genes,
screening
variants,
molecular
recording,
disease
models,
gene
therapy,
ex
therapy
potential
future
advances.
