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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.

The 
CRISPR
 Mechanism


CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) 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 ^{[2], [10]}, 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
 (Diagram 1) ^[4]. 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
 (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
 (Diagram 3).