The
increasing
prevalence
of
multi-drug-resistant
(MDR)
bacterial
pathogens
presents
a
critical
global
health
threat,
highlighting
the
urgent
need
for
innovative
approaches
to
understanding
pathogenesis
and
developing
effective
therapies.
This
review
underscores
potential
synthetic
biology
in
elucidating
host–pathogen
interactions
facilitating
creation
advanced
diagnostic
tools
targeted
therapies
combat
MDR
infections.
We
first
explore
CRISPR-based
strategies
that
modulate
essential
gene
expression,
providing
insights
into
molecular
mechanisms
underlying
interactions.
Next,
we
discuss
engineered
microbial
circuits
rapid
pathogen
detection
by
identifying
signatures
involved
interspecies
communication
swift
elimination.
Additionally,
phage
therapy
(PT),
which
leverages
bacteriophages
selectively
target
eliminate
specific
pathogens,
presenting
promising
approach
Finally,
application
organ-on-a-chip
(OOAC)
technology,
overcomes
limitations
animal
models
predicting
human
immune
responses
using
microfluidic
devices
simulate
organ-level
physiology
pathophysiology,
thereby
enabling
more
accurate
disease
modeling,
drug
testing,
development
personalized
medicine.
Collectively,
these
provide
transformative
interactions,
advancing
precise
therapeutic
against
Cell,
Год журнала:
2021,
Номер
184(17), С. 4579 - 4592.e24
Опубликована: Июль 22, 2021
Antibacterial
agents
target
the
products
of
essential
genes
but
rarely
achieve
complete
inhibition.
Thus,
all-or-none
definition
essentiality
afforded
by
traditional
genetic
approaches
fails
to
discern
most
attractive
bacterial
targets:
those
whose
incomplete
inhibition
results
in
major
fitness
costs.
In
contrast,
gene
"vulnerability"
is
a
continuous,
quantifiable
trait
that
relates
magnitude
effect
on
fitness.
We
developed
CRISPR
interference-based
functional
genomics
method
systematically
titrate
expression
Mycobacterium
tuberculosis
(Mtb)
and
monitor
outcomes.
identified
highly
vulnerable
various
processes,
including
novel
targets
unexplored
for
drug
discovery.
Equally
important,
we
invulnerable
genes,
potentially
explaining
failed
discovery
efforts.
Comparison
vulnerability
between
reference
hypervirulent
Mtb
isolate
revealed
conservation
differential
can
predict
antibacterial
susceptibility.
Our
quantitatively
redefine
processes
identify
high-value
development.
PLoS Genetics,
Год журнала:
2018,
Номер
14(11), С. e1007749 - e1007749
Опубликована: Ноя. 7, 2018
High-throughput
genetic
screens
are
powerful
methods
to
identify
genes
linked
a
given
phenotype.
The
catalytic
null
mutant
of
the
Cas9
RNA-guided
nuclease
(dCas9)
can
be
conveniently
used
silence
interest
in
method
also
known
as
CRISPRi.
Here,
we
report
genome-wide
CRISPR-dCas9
screen
using
starting
pool
~
92,000
sgRNAs
which
target
random
positions
chromosome
E.
coli.
To
benchmark
our
method,
first
investigate
its
utility
predict
gene
essentiality
genome
coli
during
growth
rich
medium.
We
could
79%
previously
reported
essential
and
demonstrate
non-essentiality
some
annotated
essential.
In
addition,
took
advantage
intermediate
repression
levels
obtained
when
targeting
template
strand
show
that
cells
very
sensitive
expression
level
limited
set
genes.
Our
data
visualized
on
CRISPRbrowser,
custom
web
interface
available
at
crispr.pasteur.fr.
then
apply
discover
required
by
phages
λ,
T4
186
kill
their
host,
highlighting
involvement
diverse
host
pathways
infection
process
three
tested
phages.
colanic
acid
capsule
synthesis
shared
resistance
mechanism
all
Finally,
plasmid
packaging
system
transduction
assay,
for
formation
functional
λ
capsids,
thus
covering
entire
phage
cycle.
This
study
demonstrates
usefulness
convenience
pooled
bacteria
paves
way
broader
use
tool
bacterial
genomics.
Cell Reports,
Год журнала:
2021,
Номер
37(5), С. 109930 - 109930
Опубликована: Ноя. 1, 2021
Mechanistic
insights
into
the
role
of
human
microbiome
in
predisposition
to
and
treatment
disease
are
limited
by
lack
methods
precisely
add
or
remove
microbial
strains
genes
from
complex
communities.
Here,
we
demonstrate
that
engineered
bacteriophage
M13
can
be
used
deliver
DNA
Escherichia
coli
within
mouse
gastrointestinal
(GI)
tract.
Delivery
a
programmable
exogenous
CRISPR-Cas9
system
enables
strain-specific
depletion
fluorescently
marked
isogenic
during
competitive
colonization
genomic
deletions
encompass
target
gene
mice
colonized
with
single
strain.
Multiple
mechanisms
allow
E.
escape
targeting,
including
loss
CRISPR
array
even
entire
system.
These
results
provide
robust
experimentally
tractable
platform
for
editing,
foundation
refinement
this
approach
increase
targeting
efficiency,
proof
concept
extension
other
phage-bacterial
pairs
interest.
Cell Systems,
Год журнала:
2020,
Номер
11(5), С. 523 - 535.e9
Опубликована: Окт. 19, 2020
Essential
genes
are
the
hubs
of
cellular
networks,
but
lack
high-throughput
methods
for
titrating
gene
expression
has
limited
our
understanding
fitness
landscapes
against
which
their
levels
optimized.
We
developed
a
modified
CRISPRi
system
leveraging
predictable
reduction
in
efficacy
imperfectly
matched
sgRNAs
to
generate
defined
activity
and
demonstrated
its
broad
applicability.
Using
libraries
mismatched
predicted
span
full
range
knockdown
levels,
we
characterized
expression-fitness
relationships
most
essential
Escherichia
coli
Bacillus
subtilis.
find
that
these
vary
widely
from
linear
bimodal
similar
within
pathways.
Notably,
despite
∼2
billion
years
evolutionary
separation
between
E.
B.
subtilis,
homologs
have
with
rare
informative
differences.
Thus,
may
reflect
homeostatic
or
constraints
shared
two
organisms.
Molecular Systems Biology,
Год журнала:
2018,
Номер
14(3)
Опубликована: Март 1, 2018
Article8
March
2018Open
Access
Source
DataTransparent
process
Tuning
dCas9's
ability
to
block
transcription
enables
robust,
noiseless
knockdown
of
bacterial
genes
Antoine
Vigouroux
Synthetic
Biology
Laboratory,
Institut
Pasteur,
Paris,
France
Microbial
Morphogenesis
and
Growth
Search
for
more
papers
by
this
author
Enno
Oldewurtel
orcid.org/0000-0002-2813-0259
Lun
Cui
orcid.org/0000-0001-5907-0538
David
Bikard
Corresponding
Author
[email
protected]
orcid.org/0000-0002-5729-1211
Sven
van
Teeffelen
orcid.org/0000-0002-0877-1294
Information
Vigouroux1,2,
Oldewurtel2,
Cui1,
*,1
*,2
1Synthetic
2Microbial
*Corresponding
author.
Tel:
+33
1
45
61
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E-mail:
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16;
Molecular
Systems
(2018)14:e7899https://doi.org/10.15252/msb.20177899
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Info
Abstract
Over
past
few
years,
tools
that
make
use
Cas9
nuclease
have
led
many
breakthroughs,
in
control
gene
expression.
The
catalytically
dead
variant
known
as
dCas9
can
be
guided
small
RNAs
target
genes,
strategy
also
CRISPRi.
Here,
we
reveal
level
complementarity
between
guide
RNA
controls
rate
at
which
polymerase
“kicks
out”
from
completes
transcription.
We
mechanism
precisely
robustly
reduce
expression
defined
relative
amounts.
Alternatively,
tuning
repression
changing
concentration
is
noisy
promoter-strength
dependent.
demonstrate
broad
applicability
method
study
genetic
regulation
cellular
physiology.
First,
characterize
feedback
strength
model
auto-repressor.
Second,
impact
amount
variations
cell-wall
synthesizing
enzymes
on
cell
morphology.
Finally,
multiplex
system
obtain
any
combination
fractional
two
genes.
Synopsis
When
encounters
inactivated
nuclease,
it
has
certain
probability
going
through
depending
sequence.
This
property
exploited
multiple
their
native
locus.
For
high
enough
concentration,
locus
saturated
only
depends
RNAP
passage
probability.
Imperfect
allows
fine-tuning
consequently
levels.
In
saturating
conditions,
does
not
produce
additional
noise
applied
measure
response
circuit,
analyze
how
synthesis
affect
shape,
tune
levels
independently.
Introduction
A
powerful
way
investigate
bacteria
vary
cell.
To
end,
are
typically
placed
under
inducible
promoters.
While
easy
implement,
approach
disadvantages:
lie
outside
dynamic
range
promoter.
promoters
increase
comparison
with
(Elowitz
et
al,
2002).
And
third,
orthogonal
systems
exist,
thus
making
multiplexing
difficult.
Recently,
different
strategies
been
devised
knock
down
amounts
levels:
Specifically,
antisense
manner
(Brophy
Voigt,
2016).
works
well
moderate
promoter
strength,
becomes
less
efficient
stronger
As
an
alternative
strategy,
knocked
varying
degrees
using
CRISPR
technology
(Bikard
2013;
Qi
2013).
catalytic
mutant
form
RNA-guided
Streptococcus
pyogenes
(dCas9)
easily
programmed
bind
position
interest
chromosome,
requirement
“NGG”
protospacer
adjacent
motif
(PAM).
unable
cleave
DNA,
but
still
binds
DNA
strongly.
If
chosen
downstream
promoter,
serves
roadblock
blocks
elongation.
single-cell
level,
interesting
implications
immune
system.
then
develop
precise
noise-preserving
independent
strength.
Target
search
begins
probing
presence
PAM
followed
melting
complementarity-dependent
strand
invasion
(Sternberg
2014;
Szczelkun
2014).
PAM-proximal
region
seed
sequence
important
binding,
several
mismatches
PAM-distal
tolerated
demonstrated
binding
assays
(Kuscu
Wu
2014)
monitoring
target-gene
Eschericha
coli
degree
controlled
quantitatively
ways:
first,
impacts
DNA.
recently
Bacillus
subtilis
where
was
xylose-inducible
(Peters
2016),
E.
strain
modified
enable
tunable
PBAD
(Li
2016);
second,
introducing
perfectly
matched
leads
very
strong
repression,
decreasing
progressively
reduces
compare
these
characterizing
properties
dCas9-mediated
level.
us
propose
novel
physical
repression.
It
previously
assumed
decreased
would
decrease
virtue
reduced
occupancy
(Farasat
Salis,
mechanism:
inside
open
reading
frame
(ORF),
determines
(RNAP)
kicks
out
during
attempt,
while
spontaneous
unbinding
negligibly
small.
saturate
target,
alone
desirable
properties:
add
extrinsic
On
contrary,
inherently
target.
complementarity-based
fluorescent-protein
reporters
inserted
upstream
its
reporter
fusions
rather
than
direct
targeting
yields
predictable
fold
characterized
provides
monitor
single
cells.
versatility
our
examples:
accurate
quantify
auto-repressor
measuring
much
actual
differs
rate.
take
advantage
steady-state
growth
operon
coding
essential
“rod”
complex,
PBP2
RodA.
multiplexed
Results
Varying
RNA-target
controlling
without
addition
CRISPR-dCas9
modulates
integrated
cassettes
constitutively
expressed
reporters,
sfgfp
superfolder
green
fluorescent
protein
(GFP)
mCherry
red
(RFP)
chromosomal
loci
MG1655.
repress
either
knock-down,
dcas9
S.
Ptet
anhydrotetracycline
(aTc)
(Qi
GFP-
RFP-coding
ORFs
array
necessary
tracrRNA,
complex
together
(Hsu
Inducing
setup
did
(Appendix
Fig
S1).
measured
stability
over
time
saw
5
days
culture.
Once
stopped
induction
all
40
clones
tested
recovered
stable,
show
toxicity.
system,
tuned
modulating
aTc
concentrations
or
spacer
numbers
5′
side
spacer.
employed
GFP
high-throughput
microscopy
(Fig
1A
B).
expected,
average
increasing
complementarity.
However,
distributions
differed
significantly
modes
modulation
1C
D).
large
cell-to-cell
fluctuations
intermediate
regime,
strongly
1D
Appendix
S2).
induced,
non-repressed
constitutive
were
recovered.
expression,
presumably
site
elaborated
below.
inducing
constant
100
ng/ml
maintained
(standard
deviation
mean)
almost
plateau
value
about
0.3
(corresponding
30%)
similar
measurements
made
others
wild-type
(Taniguchi
2010).
Complementarity-based
qualitatively
transcriptional
repressors.
example,
Lac
repressor
part
targets
fivefold
compared
unrepressed
case
(see
S3),
agreement
previous
Accordingly,
observed
if
modulated
inducer
concentration.
proposed
here
precision
Figure
1.
modulate
generating
A,
B.
Average
obtained
(A)
(B)
inducer.
Relative
given
relatively
non-targeting
Individual
points
represent
replicates.
Horizontal
bars
median
three
C,
D.
Distribution
each
experiment
panels
(A
Curves
same
color
replicates
condition.
Mechanistic
dCas9-targeted
product
probabilities:
P(stop)
blocking
upon
collision
occupying
(termed
occupancy).
determined
kon,
[dCas9],
kout.
kout,
turn,
sum
transcription-independent
kick-out
due
4
details).
F.
schematically
illustrate
behavior
(left)
(right),
respectively.
data
available
online
figure.
Data
[msb177899-sup-0002-SDataFig1.zip]
Download
figure
PowerPoint
transcribe
dCas9-bound
lack
suggested
might
test
hypothesis,
fraction
active
complexes
roughly
factor
decoy
2A).
population-averaged
flow
cytometry.
Indeed,
found
absence
both
low
2B),
confirming
hypothesis
20
11
bp.
remained
true
even
spacers
array,
regardless
S4).
effectiveness
confirmed
gradually
lowering
until
transition
no
happened
higher
decoys
one
S4B),
complex.
cases,
induction,
residual
reached
around
3%,
corresponding
concentration-independent
regime.
note
following
population
averages
performed
cytometry
generally
noisier
results
presented
2.
mismatched
depend
Left:
Schematic
assay
used
dependence
R20
RFP
perfect
match.
R11
bp
C
Introducing
acts
halves
Right:
Northern
blot
measurement
processed
R20,
reflecting
carrying
moment
measurement.
Error
standard
deviations
biological
a.u.:
arbitrary
units.
Flow
levels,
point
representing
replicate.
values
normalized
respect
(C-C).
Expression
differ
(C-R20
vs.
R20-R20,
P-value:
0.68),
nor
when
order
reversed
R20-C,
0.21),
(C-R11
R11-R11,
0.53).
P-values
come
two-sided
Student's
t-test
natural
logarithms
mean
(significance
threshold:
0.017
after
Bonferroni
correction).
2
[msb177899-sup-0003-SDataFig2.zip]
Previously,
thought
solely
is,
rates
unbinding.
According
simple
view,
should
lead
equilibrium
occupancy,
fully
occupied.
view
clear
contradiction
independence
2B).
suggests
dCas9,
bound
times,
must
responsible
reconcile
robustness
hypothesize
possible
suggest
≤
1E).
=
1,
efficiently
every
collide.
At
opposite
extreme,
0,
never
1F).
mechanism,
γ0
P(bound)
Therefore,
close
saturated.
longer
explaining
Interestingly,
moved
ORF
region,
increased
S5).
finding
pass
occupied
thanks
processive
activity,
cannot
relies
diffusion.
know
whether
influence
definition,
Repression
measurably
affected
strengths,
verify
prediction,
put
strengths
(P127
PPhlf)
blocked
four
number
mismatches.
PPhlF
times
P127
3A),
regard
promoter's
initial
identical
3B).
12
weaker
PLac
mM
IPTG
S6).
These
observations
confirm
conditions
3.
conditionsRelative
repressed
set
(A,
B)
non-saturating
(C)
concentrations.
Raw
P127,
C.
Experimental
predicted
(using
lower
aTc).
PPhlf
up
six
RNA,
quantitative
Appendix).
bars:
error
computational
prediction.
3
[msb177899-sup-0004-SDataFig3.zip]
supporting
“kick-out”
ejection
successful
wondered
ejected
events.
displacements
they
could
RNAP,
kon[dCas9]
kout
combined
RNAP-induced
ejections,
possibly
replication-fork-based
(Jones
2017;
see
therefore
P(bound),
which,
fold.
observation
agrees
full
(14
bp)
complementarity,
respectively
3C
S7A).
predicts
dominated
events,
rare
(4;
S7B).
compatible
leaves
spontaneously
gets
kicked
replication.
prediction
consistent
long
half-life
reported
vivo
2017)
vitro
expected
valid
14
bp,
1A)
remains
become
equally
collision-based
ejections
below
some
Yet,
applications
below,
rebinding
paragraph).
proportional
rate,
principle
events
reads
template
increases
temperature
(Wiktor
2016)
42°C,
suggesting
temperature.
bears
possibility
robust
dCas9-copy
temperature,
condition
saturation
fulfilled.
robustness,
temperatures
ranging
30
42°C.
decayed
continuously
4),
displaying
sharp
37
Regardless
S8).
From
model,
conclude
being
RNAP.
indicates
work
independently
tested.
4.
efficiency
temperaturesRelative
temperatures.
[msb177899-sup-0005-SDataFig4.zip]
insertions
contexts
Precision,
versatile
strategy.
context,
insert
translational
gene.
provide
convenient
CRISPR-based
perform
inspired
allelic
exchange
(Pósfai
1999)
Text
S9).
library
plasmids
introduced
desired
sequences.
taking
established
above.
Furthermore,
revealing
variations.
addgene
(https://www.addgene.org/depositor-collections/bikard-crispr-repression/).
following,
physiology:
effect
morphology
growth.
uncover
circuits,
chose
described
PhlF
auto-repres
Nature Communications,
Год журнала:
2019,
Номер
10(1)
Опубликована: Авг. 7, 2019
CRISPR-Cas9
is
widely
used
in
genomic
editing,
but
the
kinetics
of
target
search
and
its
relation
to
cellular
concentration
Cas9
have
remained
elusive.
Effective
requires
constant
screening
protospacer
adjacent
motif
(PAM)
a
30
ms
upper
limit
for
was
recently
found.
To
further
quantify
rapid
switching
between
DNA-bound
freely-diffusing
states
dCas9,
we
developed
an
open-microscopy
framework,
miCube,
introduce
Monte-Carlo
diffusion
distribution
analysis
(MC-DDA).
Our
reveals
that
dCas9
PAMs
40%
time
Gram-positive
Lactoccous
lactis,
averaging
17
±
4
per
binding
event.
Using
heterogeneous
expression,
determine
number
target-containing
plasmids
derive
copy
dependent
cleavage.
Furthermore,
show
not
irreversibly
bound
sites
can
still
interfere
with
plasmid
replication.
Taken
together,
our
quantitative
data
facilitates
optimization
CRISPR-Cas
toolbox.
Philosophical Transactions of the Royal Society B Biological Sciences,
Год журнала:
2019,
Номер
374(1772), С. 20180103 - 20180103
Опубликована: Март 25, 2019
Our
bodies
are
colonized
by
a
complex
ecosystem
of
bacteria,
unicellular
eukaryotes
and
their
viruses
that
together
play
major
role
in
our
health.
Over
the
past
few
years
tools
derived
from
prokaryotic
immune
system
known
as
CRISPR-Cas
have
empowered
researchers
to
modify
study
organisms
with
unprecedented
ease
efficiency.
Here
we
discuss
how
various
types
systems
can
be
used
genome
gut
microorganisms
bacteriophages.
also
delivered
bacterial
population
programmed
specifically
eliminate
members
microbiome.
Finally,
engineered
control
gene
expression
modulate
production
metabolites
proteins.
Together
these
provide
exciting
opportunities
investigate
interplay
between
microbiome
bodies,
present
new
avenues
for
development
drugs
target
This
article
is
part
discussion
meeting
issue
‘The
ecology
evolution
adaptive
systems’.