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| | {{:Team:Arizona_State/Templates/sidebar|title=Project}} | | {{:Team:Arizona_State/Templates/sidebar|title=Project}} |
| - | * Our project will have several stages, all pursuant to the general investigation and modularization of the CRISPR pathway:
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| - | :* Proof of concept targeting reporters such as GFP, eventually creating a CRISPR biobrick
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| - | :* Investigate CRISPR system dynamics based on factors such as degradation of self-targeting sequences and maintenance of the array.
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| - | :* Target genes such as NDM-1 or other clinically relevant pathways.
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| - |
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| - | '''NDM-1 in
Perspective
'''
| |
| - | ----
| |
| - | <p>Global
antibiotic
resistance
is
a
concern
of
the
utmost
importance
to
the
World
Health
Organization
and
health care
everywhere.
Bacteria
that
have
acquired
antibiotic
resistance
jeopardize
world
health care
as
a
whole,
because
they
increase
mortality
rate
of
normally
curable
infections,
and
there
is
no
coherent
approach
to
containing
and
countering
resistant
strains.
New
Delhi
Metallo‐Beta‐Lactamse
(NDM‐1)
containing
bacteria
are
particularly
ominous
because
the
NDM‐1
enzyme
hydrolyzes
a
broad
range
of
potent
beta‐lactam
antibiotics
(e.g.
carbapenems).
This
enzyme
is
effective
in
rendering
normal
lines
of
treatment
for
bacterial
infection
useless.
NDM‐1
positive
strains
originated
in
India
and
Pakistan
and
have
recently
spread
to
the
UK,
Europe,
and
Canada.
There
has
also
been
a
drastic
increase
in
the
number
of
reported
NDM‐1
positive
cases
in
the
United
States,
according
to
the
Centers
of
Disease
Control
and
Prevention.
Viable
antibiotics
as a
resource
are
becoming more
and
more
deficient.
Alternative
solutions
to
resistance
must
be
promptly
sought
and
intelligently
employed
to
counter
the
threat
of
antibiotic
resistant
bacteria.
</p>
| |
| - |
| |
| - | '''The
CRISPR
Mechanism
'''
| |
| - | ----
| |
| - | CRISPR ('''C'''lustered '''R'''egularly '''I'''nterspaced '''S'''hort '''P'''alindromic '''R'''epeats) is a genomic feature of some prokaryotes and most archea. A CRISPR locus consists of a set of CAS (CRISPR associated) genes, a leader, or promoter, sequence, and an array. This array consists of repeating elements along with "spacers". These spacer regions direct the CRISPR machinery to degrade or otherwise inactivate a complementary sequence in the cell.
| |
| - |
| |
| - |
The
CRISPR / CAS pathway
can
be
viewed
as
a
prokaryotic
immune system
or as an analogue to eukaryotic RNAi. This
mechanism
of
bacterial
survival
can in theory
be
directed
to
silence
a
gene
of
interest, which affords
us
an
interesting
method
to
tackle
the
aforementioned
problem.
CRISPR
gene
loci
have
been
demonstrated
to
equip
both
prokaryotes
and
archaea
with
a
defense
mechanism
against
exogenous
DNA
and
RNA
sequences <sup>[[Team:Arizona State/Project/References#ref2|[2]]], [[Team:Arizona State/Project/References#ref10|[10]]]</sup>, usually targeting invading viruses.
The
transcripts
from
the
spacer/repeat
region
undergo
hair
pinning
due
to
the
palindromic
sequence
structure.
The
peptide
products
of
the CAS genes
work
cooperatively
with
crRNA
to
silence
a
complimentary
target
[[#diagram1| (Diagram 1)]] <sup>[[Team:Arizona State/Project/References#ref4|[4]]]</sup>. The
function
is
a
prokaryotic
analog
to
both
RNA
interference
and
immunity.
CRISPR
presents
it self
as
a
potentially
useful
tool
in
prokaryotic
gene
manipulation.
Our
goal
as
ASU’s
first
iGEM
team
is
to
develop
a
CRISPR
plasmid
that
contains
elements
to
target
and
silence
the
NDM‐1
gene
sequence
[[#diagram2| (Diagram 2)]].
While our final goal is the
targeting of
NDM‐1,
we
recognize
that
CRISPR can
potentially
target
any
gene
of
interest.
We
will
develop
a
robust, modular
platform
for
gene
silencing based on CRISPR.
The
final
product
of
this
project
will
be
a
fully
functioning
CRISPR
array
that
will
be
submitted
to
the
Standard
Registry
of
Biological
Parts,
an
open‐source
collection
of
DNA
building
blocks,
as
a
BioBrick,
a
modular
component
for
genetic
engineering
[[#diagram3| (Diagram 3)]].
| |
| - |
| |
| - | <div id="diagram1"></div>
| |
| - | [[Image:Arizona_State_proposal_diagram1.png|center]]
| |
| - |
| |
| - | <div id="diagram2"></div>
| |
| - | [[Image:Arizona_State_proposal_diagram2.png|center]]
| |
| - |
| |
| - |
| |
| - | <div id="diagram3"></div>
| |
| - | [[Image:Arizona_State_proposal_diagram3.png|center]]
| |
"
