the application of crispr/cas9 system in genome editing

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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