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The application of CRISPR/Cas9 system in
genome editing
By: Arash Zolnori
Supervisor: Dr. tavakol
Introduction
CRISPR C: ClusteredR: regularly
I: interspaced S: short P: palindromic R: repeat
The CRISPR system
- based on bacterial immune system
- normally occurring in bacterial processes
- first reported in 1987 for the Ecoli But at the same time
Their function was unknown
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The CRISPR system
-In 2000 similar repeats were identified in other bacteria
- In 2002 similar repeats were named CRISPR and a
set of proteins was found to be associated with CRISPR
repeats (Cas: CRISPR associated gene)
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The CRISPR system
- In 2005 research showed that some CRISPR spacers
are derived from bacteriophage
- In 2007 researcher could use spacer DNA to alter the
resistance of s.thermophilus to phage attack
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The CRISPR system
and finally in 2015 :
Doudna and Charpentier discovered that how bacteria
use spacers in it’s immune system
Jennifer Doudna Emmanuelle Charpentier4
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Stage 1: CRISPR spacer acquisition
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Stage 2: CRISPR expression
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Stage 3: interference
Interference system rely on :
-association of Cas protein with sgRNAs and making
ribonucleoprotein Complex (RNP)
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Stage 3: interference
11RNP Complex
Stage 3: interference
Interference system rely on :
- PAM: protospacer adjacent motifs
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Stage 3: interference
5'-NGG-3'
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Stage 3: interference
- Cas is an endonuclease enzyme which have two
cleavage domain (DSB)
- Viral target DNA will cleaving at 3 bp on PAM
upstream
- Multiple viral spacer acquisition = Multiple cleavage
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Application of CRISPR/Cas9 in genome editing
- Endonucleases are usually 4-8 bp cutter
- Animal and plant DNA length is around
1000-3000 Mb
- So it’s will make a lot of unwanted band
(digestion produces)
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- Cas-sgRNA complex is a smart and
programmable Nuclease
As stated before :
- RNP recognition sites is 17-20 nt
(spacer lenght)
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Cas-sgRNA complex can:
- Make a DSB in any desired target location
- So CRISPR system have a great potential to become a
genome editing tool
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HOW ?First : we have to designing spacer
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- tracrRNA : trans-activating CRISPR RNA
- crRNA : CRISPR RNA
dsRNA
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principles
- Minimum off-target- GC content: the typical range is between
40% - 80% - Online sites for CRISPR designing
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CRISPR designing
Online sites:- http://crispr.mit.edu/
- http://crispr.u-psud.fr/Server/
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second : Cas9 principles
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Streptococcus pyogenes Cas9 :
-the standard cas9 used in researches
- PAM seq : 5’ NGG 3’
Staphylococcus aureus Cas9 :- Smaller than s.pyogenes Cas9
- PAM seq : 5’ NNGRRT 3’
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In term of cleavage :
- There is no different between cds or non-cds
locations and this is one of CRISPR/Cas9
advantages
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third : what is our purpose to making a DSB in target DNA
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Knock in
HDR :
homology
directed
repair
Knock out
NHEJ:
non
homologous
end joining
or
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Gene of Interest
Protospacer Adjacent Motif (PAM)
Target Sequence
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Non-Homologous End Joining (NHEJ)
and DNA repair pathway30
Transferring crRNA:tracrRNA-Cas9 complex to the cell nucleus
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Non – viral based gene delivery :
- Lipid-Mediated Transfection
- Calcium phosphate transfection
- Electroporation
ghfgh
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viral based gene delivery :
- Lentivirus
-Adenovirus
-Adeno-Associated Virus (AAV)
- crispr plasmid (pCRISPR)
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Correction of Mutations in Zygote stages of Human?
We have more knowledge and techniques on Human Embryo than Monkey’s36
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pX K7 -AtCa s 9 -214488 bp
Eg fp
co m p lem en tary
co m p le 2
N L S-At
N L S-At
C as9
at t B 1
att B 2
K an
Sm /Sp R
p35S
T 35S
L B
R B
3X F L AG
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conclusion
Application of CRISPR/Cas9
- Knockout/Knockin Mouse generation :
Traditional methods: at least 6~12 monthsCRISPR/Cas9 : 2 Months with ~90% of efficiency
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- with cas9 nickase ability off-target effects will reducing to 0%- there is no need to backcrossing F1 lines with parent lines- Site-directed mutagenesis: Disease Model Generation-Curing genetic diseases like HIV and Cancer-Gene Activation / Repression by dCas9- It’s changes is inheritable- low price
In Here, donor strand is a ssDNA
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