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Project

• Our project will have several stages, all pursuant to the general investigation and modularization of the CRISPR pathway:

• Proof of concept targeting reporters such as GFP, eventually creating a CRISPR biobrick
• Investigate CRISPR system dynamics based on factors such as degradation of self-targeting sequences and maintenance of the array.
• Target genes such as NDM-1 or other clinically relevant pathways.

NDM­-1 in
 Perspective


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.


The 
CRISPR
 Mechanism



The 
CRISPR‐Cas
 pathway 
can 
be
 compared 
to
 a
 prokaryotic
 immunity 
or 
RNA 
interference
 that
 can 
be 
directed 
to 
silence 
a
gene 
of 
interest.
 This 
mechanism
 of
 bacterial
 survival
 affords
 us
 an 
interesting 
method 
to 
tackle 
the 
aforementioned
problem. 
Clustered 
Regularly
 Interspaced
 Short 
Palindromic
 Repeats
(CRISPR)
 gene 
loci 
have 
been
 demonstrated
 to
 equip
 both 
prokaryotes
 and 
archaea 
with 
a 
defense
 mechanism
 against 
exogenous
 DNA
 and 
RNA 
sequences [1], [2]. 
CRISPR
 genes
 appear 
in 
an
 array 
that 
contains 
contiguous 
spacers, 
repeats, 
and 
an 
operon 
of 
structural
 genes. 
The
 transcripts 
from 
the 
spacer/repeat 
region 
undergo 
hair
pinning 
due 
to 
the 
palindromic
 sequence
 structure. 
The 
peptide
 products 
of 
the 
CRISPR‐associated 
structural
 genes 
(CAS) 
work
 cooperatively 
with 
crRNA 
to 
silence 
a 
complimentary 
target
 (Diagram
1) [3]. The 
function 
is 
a
 prokaryotic
 analog 
to 
both 
RNA 
interference 
and 
immunity. 
CRISPR
 quickly 
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
 (Diagram
2). 
While 
targeting 
NDM‐1,
 we 
recognize 
that 
CRISPR can
 potentially 
target 
any 
gene 
of
 interest,
 thus 
we 
will 
develop 
a
robust 
platform 
for 
gene 
silencing. 
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
 (Diagram
3).


CRISPR / CAS / RAMP Overview

References