Nature Biotechnology,
Год журнала:
2022,
Номер
41(1), С. 96 - 107
Опубликована: Сен. 8, 2022
Despite
the
availability
of
Cas9
variants
with
varied
protospacer-adjacent
motif
(PAM)
compatibilities,
some
genomic
loci-especially
those
pyrimidine-rich
PAM
sequences-remain
inaccessible
by
high-activity
proteins.
Moreover,
broadening
sequence
compatibility
through
engineering
can
increase
off-target
activity.
With
directed
evolution,
we
generated
four
that
together
enable
targeting
most
sequences
in
human
genome.
Using
phage-assisted
noncontinuous
evolution
and
eVOLVER-supported
continuous
evolved
Nme2Cas9,
a
compact
variant,
into
recognize
single-nucleotide
pyrimidine-PAM
sequences.
We
developed
general
selection
strategy
requires
functional
editing
fully
specified
target
protospacers
PAMs.
applied
this
to
evolve
eNme2-T.1,
eNme2-T.2,
eNme2-C
eNme2-C.NR.
Variants
eNme2-T.1
eNme2-T.2
offer
access
N
Heliyon,
Год журнала:
2024,
Номер
10(4), С. e26179 - e26179
Опубликована: Фев. 1, 2024
CRISPR/Cas
systems
have
become
integral
parts
of
nucleic
acid
detection
apparatus
and
biosensors.
Various
such
as
CRISPR/Cas9,
CRISPR/Cas12,
CRISPR/Cas13,
CRISPR/Cas14
CRISPR/Cas3
utilize
different
mechanisms
to
detect
or
differentiate
biological
activities
nucleotide
sequences.
Usually,
CRISPR/Cas-based
are
combined
with
polymerase
chain
reaction,
loop-mediated
isothermal
amplification,
recombinase
amplification
transcriptional
technologies
for
effective
diagnostics.
Premised
on
these,
many
biosensors
been
developed
acids
viral
bacterial
pathogens
in
clinical
samples,
well
other
applications
life
sciences
including
biosecurity,
food
safety
environmental
assessment.
Additionally,
showed
better
specificity
compared
molecular
diagnostic
methods.
In
this
review,
we
give
an
overview
various
methods
highlight
some
advances
their
development
components.
We
also
discourse
operational
challenges
advantages
disadvantages
systems.
Finally,
important
considerations
offered
the
improvement
testing.
Directed
protein
evolution
is
central
to
biomedical
applications
but
faces
challenges
like
experimental
complexity,
inefficient
multi-property
optimization,
and
local
maxima
traps.
While
in
silico
methods
using
language
models
(PLMs)
can
provide
modeled
fitness
landscape
guidance,
they
struggle
generalize
across
diverse
families
map
activity.
We
present
EVOLVEpro,
a
few-shot
active
learning
framework
that
combines
PLMs
regression
rapidly
improve
EVOLVEpro
surpasses
current
methods,
yielding
up
100-fold
improvements
desired
properties.
demonstrate
its
effectiveness
six
proteins
RNA
production,
genome
editing,
antibody
binding
applications.
These
results
highlight
the
advantages
of
with
minimal
data
over
zero-shot
predictions.
opens
new
possibilities
for
AI-guided
engineering
biology
medicine.
Nature Communications,
Год журнала:
2025,
Номер
16(1)
Опубликована: Янв. 15, 2025
Abstract
With
recent
advancements
in
gene
editing
technology
using
the
CRISPR/Cas
system,
there
is
a
demand
for
more
effective
editors.
A
key
factor
facilitating
efficient
CRISPR
delivery
into
cells,
which
known
to
be
associated
with
size
of
system.
Accordingly,
compact
CRISPR-Cas
systems
derived
from
various
strains
are
discovered,
among
Un1Cas12f1
2.6
times
smaller
than
SpCas9,
providing
advantages
therapy
research.
Despite
extensive
engineering
efforts
improve
Un1Cas12f1,
efficiency
still
shown
low
depending
on
target
site.
To
overcome
this
limitation,
we
develop
enhanced
Cas12f1
(eCas12f1),
exhibits
activity
similar
SpCas9
and
AsCpf1,
even
targets
where
previously
improved
variants
showed
efficiency.
Furthermore,
demonstrate
that
eCas12f1
efficiently
induces
apoptosis
cancer
cells
compatible
base
regulation
expression,
verifying
its
high
utility
applicability
Nature Biotechnology,
Год журнала:
2022,
Номер
41(1), С. 96 - 107
Опубликована: Сен. 8, 2022
Despite
the
availability
of
Cas9
variants
with
varied
protospacer-adjacent
motif
(PAM)
compatibilities,
some
genomic
loci-especially
those
pyrimidine-rich
PAM
sequences-remain
inaccessible
by
high-activity
proteins.
Moreover,
broadening
sequence
compatibility
through
engineering
can
increase
off-target
activity.
With
directed
evolution,
we
generated
four
that
together
enable
targeting
most
sequences
in
human
genome.
Using
phage-assisted
noncontinuous
evolution
and
eVOLVER-supported
continuous
evolved
Nme2Cas9,
a
compact
variant,
into
recognize
single-nucleotide
pyrimidine-PAM
sequences.
We
developed
general
selection
strategy
requires
functional
editing
fully
specified
target
protospacers
PAMs.
applied
this
to
evolve
eNme2-T.1,
eNme2-T.2,
eNme2-C
eNme2-C.NR.
Variants
eNme2-T.1
eNme2-T.2
offer
access
N
