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Project

Overall 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
itself
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


1. Barrangou 
R,
 Fremaux
 C,
 Deveau 
H, 
Richards 
M, 
Boyaval
 P, 
Moineau 
S,
 Romero 
D, 
Horvath 
P. 
CRISPR 
Provides 
Acquired
 Resistance 
Against 
Viruses 
in
 Prokaryotes. 
Science 
315, 
1709‐1712
 (2007).

2. Karginov
 FV,
 Hannon 
GJ. 
The 
CRISPR 
system: 
small 
RNA‐guided
 defense 
in 
bacteria 
and 
archaea. 
Molecular 
Cell 
37,
7‐19
(2010).

3. Brouns 
SJ, 
Jore 
MM, 
Lundgren 
M, 
Westra 
ER, 
Slijkhuis 
RJ,
 Snijders
 AP, 
Dickman 
MJ,
 Makarova 
KS,
 Koonin
 EV, 
van
der
 Oost 
J.
 Small 
CRISPR 
RNAs 
guide 
antiviral 
defense 
in
 prokaryotes.
 Science 
321, 960‐964 
(2008).