Frontiers in Plant Science,
Год журнала:
2023,
Номер
13
Опубликована: Янв. 10, 2023
The
advancement
of
precision
engineering
for
crop
trait
improvement
is
important
in
the
face
rapid
population
growth,
climate
change,
and
disease.
To
this
end,
targeted
double-stranded
break
technology
using
RNA-guided
Cas9
has
been
adopted
widely
genome
editing
plants.
Agrobacterium
or
particle
bombardment-based
delivery
plasmids
encoding
guide
RNA
(gRNA)
common,
but
requires
optimization
expression
often
results
random
integration
plasmid
DNA
into
plant
genome.
Recent
advances
have
described
gene
by
gRNA
as
pre-assembled
ribonucleoproteins
(RNPs)
various
tissues,
with
moderate
efficiency
resulting
regenerated
In
report
we
describe
significant
improvements
to
Cas9-RNP
mediated
wheat.
We
demonstrate
that
assays
protoplasts
are
a
fast
effective
tool
rational
selection
optimal
gRNAs
regenerable
immature
embryos
(IEs),
high
temperature
treatment
enhances
rates
both
tissue
types.
also
show
Cas9-mediated
persists
at
least
14
days
gold
bombarded
wheat
IEs.
edited
plants
work
recovered
absence
exogenous
selection.
With
method,
produce
knockouts
set
three
homoeologous
genes
two
pathogenic
effector
susceptibility
genes,
insensitivity
corresponding
necrotrophic
effectors
produced
Parastagonospora
nodorum.
establishment
highly
efficient,
DNA-free
holds
promise
accelerated
diversity
production
an
expansive
array
crops.
Journal of Advanced Research,
Год журнала:
2024,
Номер
unknown
Опубликована: Авг. 1, 2024
Soybean
is
a
worldwide-cultivated
crop
due
to
its
applications
in
the
food,
feed,
and
biodiesel
industries.
Genome
editing
soybean
began
with
ZFN
TALEN
technologies;
however,
CRISPR/Cas
has
emerged
shortly
became
preferable
approach
for
genome
manipulation
since
it
more
precise,
easy
handle,
cost-effective.
Recent
reports
have
focused
on
conventional
Cas9
nuclease,
nickase
(nCas9)
derived
base
editors,
Cas12a
(formally
Cpf1)
as
most
commonly
used
editors
soybean.
Nonetheless,
several
challenges
complex
plant
genetic
engineering
pipeline
need
be
overcome
effectively
edit
of
an
elite
cultivar.
These
include
(1)
optimizing
CRISPR
cassette
design
(i.e.,
gRNA
Cas
promoters,
testing,
number
gRNAs,
binary
vector),
(2)
improving
transformation
frequency,
(3)
increasing
efficiency
ratio
targeted
cells,
(4)
production.
Plant Biotechnology Journal,
Год журнала:
2025,
Номер
unknown
Опубликована: Фев. 10, 2025
Summary
Transgene‐free
genome
editing
is
important
for
crop
improvement
as
it
reduces
unanticipated
genomic
changes.
While
mRNA
delivery
systems
offer
a
powerful
method
achieving
transgene‐free
editing,
they
remain
inefficient
and
challenging
in
plants.
Here
we
describe
an
efficient
system
plants
with
substantially
improved
efficiency.
By
optimizing
the
5′
untranslated
regions
(5′UTRs)
poly(A)
tails
of
vitro
‐transcribed
(IVT)
mRNAs
coating
protamine
during
particle
bombardment,
have
developed
optimized
termed
v2_TMV/DEN2.
This
enhanced
efficiencies
knock‐out,
A‐to‐G
C‐to‐T
base
by
average
4.7‐,
3.4‐
2.5‐fold
at
various
endogenous
sites
compared
plasmid‐based
transient
via
bombardment
rice
suspension
cells
wheat
immature
embryos
48
h
post‐transformation.
Furthermore,
obtained
edited
5.0–180.8%
26.1–26.2%
using
v2_TMV/DEN2
wheat,
respectively,
0.0–43.2%
4.7–10.4%
plasmids.
Our
study
provides
convenient
PLANT PHYSIOLOGY,
Год журнала:
2021,
Номер
188(4), С. 1757 - 1768
Опубликована: Дек. 7, 2021
Abstract
Transgene
residuals
in
edited
plants
affect
genetic
analysis,
pose
off-target
risks,
and
cause
regulatory
concerns.
Several
strategies
have
been
developed
to
efficiently
edit
target
genes
without
leaving
any
transgenes
plants.
Some
approaches
directly
address
this
issue
by
editing
plant
genomes
with
DNA-free
reagents.
On
the
other
hand,
DNA-based
techniques
require
another
step
for
ensuring
are
transgene-free.
Fluorescent
markers,
pigments,
chemical
treatments
all
employed
as
tools
distinguish
transgenic
from
transgene-free
quickly
easily.
Moreover,
suicide
used
trigger
self-elimination
of
plants,
greatly
improving
efficiency
isolating
desired
Transgenes
can
also
be
excised
using
site-specific
recombination,
transposition
or
gene
nucleases,
providing
a
strategy
asexually
produced
Finally,
haploid
induction
coupled
may
make
it
feasible
that
recalcitrant
transformation.
Here,
we
evaluate
strengths
weaknesses
recently
obtaining
transgene
residuals.
Frontiers in Plant Science,
Год журнала:
2023,
Номер
13
Опубликована: Янв. 10, 2023
The
advancement
of
precision
engineering
for
crop
trait
improvement
is
important
in
the
face
rapid
population
growth,
climate
change,
and
disease.
To
this
end,
targeted
double-stranded
break
technology
using
RNA-guided
Cas9
has
been
adopted
widely
genome
editing
plants.
Agrobacterium
or
particle
bombardment-based
delivery
plasmids
encoding
guide
RNA
(gRNA)
common,
but
requires
optimization
expression
often
results
random
integration
plasmid
DNA
into
plant
genome.
Recent
advances
have
described
gene
by
gRNA
as
pre-assembled
ribonucleoproteins
(RNPs)
various
tissues,
with
moderate
efficiency
resulting
regenerated
In
report
we
describe
significant
improvements
to
Cas9-RNP
mediated
wheat.
We
demonstrate
that
assays
protoplasts
are
a
fast
effective
tool
rational
selection
optimal
gRNAs
regenerable
immature
embryos
(IEs),
high
temperature
treatment
enhances
rates
both
tissue
types.
also
show
Cas9-mediated
persists
at
least
14
days
gold
bombarded
wheat
IEs.
edited
plants
work
recovered
absence
exogenous
selection.
With
method,
produce
knockouts
set
three
homoeologous
genes
two
pathogenic
effector
susceptibility
genes,
insensitivity
corresponding
necrotrophic
effectors
produced
Parastagonospora
nodorum.
establishment
highly
efficient,
DNA-free
holds
promise
accelerated
diversity
production
an
expansive
array
crops.
