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.
Proceedings of the National Academy of Sciences,
Год журнала:
2023,
Номер
120(4)
Опубликована: Янв. 18, 2023
Clustered
regularly
interspaced
short
palindromic
repeats
and
CRISPR-associated
proteins
(CRISPR-Cas)
systems
have
been
developed
as
important
tools
for
plant
genome
engineering.
Here,
we
demonstrate
that
the
hypercompact
CasΦ
nuclease
is
able
to
generate
stably
inherited
gene
edits
in
Arabidopsis
,
guide
RNAs
can
be
expressed
with
either
Pol-III
U6
promoter
or
a
Pol-II
together
ribozyme
mediated
RNA
processing.
Using
fwa
epiallele,
show
displays
higher
editing
efficiency
when
target
locus
not
DNA
methylated,
suggesting
sensitive
chromatin
environment.
Importantly,
two
protein
variants,
vCasΦ
nCasΦ,
both
showed
much
relative
wild-type
enzyme.
Consistently,
nCasΦ
yielded
offspring
plants
at
rates
compared
WTCasΦ.
Extensive
genomic
analysis
of
edited
no
off-target
editing,
highly
specific.
The
size,
T-rich
minimal
protospacer
adjacent
motif
(PAM),
wide
range
working
temperatures
make
an
excellent
supplement
existing
systems.
Cas12a
(formerly
known
as
Cpf1),
the
class
II
type
V
CRISPR
nuclease,
has
been
widely
used
for
genome
editing
in
mammalian
cells
and
plants
due
to
its
distinct
characteristics
from
Cas9.
Despite
being
one
of
most
robust
nucleases,
LbCas12a
general
is
less
efficient
than
SpCas9
human
cells,
animals,
plants.
Nucleic Acids Research,
Год журнала:
2024,
Номер
52(6), С. 3234 - 3248
Опубликована: Янв. 23, 2024
Abstract
Cas9
and
Cas12
nucleases
of
class
2
CRISPR-Cas
systems
provide
immunity
in
prokaryotes
through
RNA-guided
cleavage
foreign
DNA.
Here
we
characterize
a
set
compact
CRISPR-Cas12m
(subtype
V-M)
effector
proteins
show
that
they
protection
against
bacteriophages
plasmids
the
targeted
DNA
binding
rather
than
cleavage.
Biochemical
assays
suggest
Cas12m
effectors
can
act
as
roadblocks
inhibiting
transcription
and/or
replication,
thereby
triggering
interference
invaders.
Cryo-EM
structure
Gordonia
otitidis
(Go)
ternary
complex
provided
here
reveals
structural
mechanism
ensuring
interference.
Harnessing
GoCas12m
innate
ability
to
bind
target
fused
it
with
adenine
deaminase
TadA-8e
showed
an
efficient
A-to-G
editing
Escherichia
coli
human
cells.
Overall,
this
study
expands
our
understanding
functionally
diverse
protein
family,
revealing
DNA-binding
dependent
could
be
harnessed
for
engineering
base-editing
tools.
Molecular Cell,
Год журнала:
2022,
Номер
82(10), С. 1865 - 1877.e4
Опубликована: Апрель 1, 2022
RNA-guided
CRISPR-Cas
nucleases
are
widely
used
as
versatile
genome-engineering
tools.
Recent
studies
identified
functionally
divergent
type
V
Cas12
family
enzymes.
Among
them,
Cas12c2
binds
a
CRISPR
RNA
(crRNA)
and
trans-activating
crRNA
(tracrRNA)
recognizes
double-stranded
DNA
targets
with
short
TN
PAM.
Here,
we
report
the
cryo-electron
microscopy
structures
of
Cas12c2-guide
binary
complex
RNA-target
ternary
complex.
The
revealed
that
tracrRNA
form
an
unexpected
X-junction
architecture,
single
T
nucleotide
in
PAM
through
specific
hydrogen-bonding
interactions
two
arginine
residues.
Furthermore,
our
biochemical
analyses
indicated
processes
its
precursor
to
mature
using
RuvC
catalytic
site
unique
mechanism.
Collectively,
findings
improve
mechanistic
understanding
diverse
effectors.
Nature Communications,
Год журнала:
2022,
Номер
13(1)
Опубликована: Май 20, 2022
Abstract
The
CRISPR-Cas
type
V-I
is
a
family
of
Cas12i-containing
programmable
nuclease
systems
guided
by
short
crRNA
without
requirement
for
tracrRNA.
Here
we
present
an
engineered
Type
CRISPR
system
(Cas12i),
ABR-001,
which
utilizes
tracr-less
guide
RNA.
compact
Cas12i
effector
capable
self-processing
pre-crRNA
and
cleaving
dsDNA
targets,
facilitates
versatile
delivery
options
multiplexing,
respectively.
We
apply
unbiased
mutational
scanning
approach
to
enhance
initially
low
editing
activity
Cas12i2.
variant,
exhibits
broad
genome
capability
in
human
cell
lines,
primary
T
cells,
CD34+
hematopoietic
stem
progenitor
with
both
robust
efficiency
high
specificity.
In
addition,
ABR-001
achieves
level
when
delivered
via
AAV
vector
HEK293T
cells.
This
work
establishes
as
versatile,
specific,
high-performance
platform
ex
vivo
gene
therapy.
