48
The application of CRISPR/Cas9 system in genome editing By: Arash Zolnori Supervisor: Dr. tavakol

the application of CRISPR/Cas9 system in genome editing

Embed Size (px)

Citation preview

Page 1: the application of CRISPR/Cas9 system in genome editing

The application of CRISPR/Cas9 system in

genome editing

By: Arash Zolnori

Supervisor: Dr. tavakol

Page 2: the application of CRISPR/Cas9 system in genome editing

Introduction

Page 3: the application of CRISPR/Cas9 system in genome editing

CRISPR C: ClusteredR: regularly

I: interspaced S: short P: palindromic R: repeat

Page 4: the application of CRISPR/Cas9 system in genome editing

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

1

Page 5: the application of CRISPR/Cas9 system in genome editing

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)

2

Page 6: the application of CRISPR/Cas9 system in genome editing

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

3

Page 7: the application of CRISPR/Cas9 system in genome editing

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

Page 8: the application of CRISPR/Cas9 system in genome editing

5

Page 9: the application of CRISPR/Cas9 system in genome editing
Page 10: the application of CRISPR/Cas9 system in genome editing
Page 11: the application of CRISPR/Cas9 system in genome editing
Page 12: the application of CRISPR/Cas9 system in genome editing
Page 13: the application of CRISPR/Cas9 system in genome editing

6

Page 14: the application of CRISPR/Cas9 system in genome editing

7

Page 15: the application of CRISPR/Cas9 system in genome editing

Stage 1: CRISPR spacer acquisition

8

Page 16: the application of CRISPR/Cas9 system in genome editing

Stage 2: CRISPR expression

9

Page 17: the application of CRISPR/Cas9 system in genome editing

Stage 3: interference

Interference system rely on :

-association of Cas protein with sgRNAs and making

ribonucleoprotein Complex (RNP)

10

Page 18: the application of CRISPR/Cas9 system in genome editing

Stage 3: interference

11RNP Complex

Page 19: the application of CRISPR/Cas9 system in genome editing

Stage 3: interference

Interference system rely on :

- PAM: protospacer adjacent motifs

12

Page 20: the application of CRISPR/Cas9 system in genome editing

Stage 3: interference

5'-NGG-3'

13

Page 21: the application of CRISPR/Cas9 system in genome editing

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

14

Page 22: the application of CRISPR/Cas9 system in genome editing

15

Page 23: the application of CRISPR/Cas9 system in genome editing

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)

16

Page 24: the application of CRISPR/Cas9 system in genome editing

- Cas-sgRNA complex is a smart and

programmable Nuclease

As stated before :

- RNP recognition sites is 17-20 nt

(spacer lenght)

17

Page 25: the application of CRISPR/Cas9 system in genome editing

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

18

Page 26: the application of CRISPR/Cas9 system in genome editing

HOW ?First : we have to designing spacer

19

Page 27: the application of CRISPR/Cas9 system in genome editing

- tracrRNA : trans-activating CRISPR RNA

- crRNA : CRISPR RNA

dsRNA

20

Page 28: the application of CRISPR/Cas9 system in genome editing

21

Page 29: the application of CRISPR/Cas9 system in genome editing

principles

- Minimum off-target- GC content: the typical range is between

40% - 80% - Online sites for CRISPR designing

22

Page 30: the application of CRISPR/Cas9 system in genome editing

CRISPR designing

Online sites:- http://crispr.mit.edu/

- http://crispr.u-psud.fr/Server/

23

Page 31: the application of CRISPR/Cas9 system in genome editing

second : Cas9 principles

24

Page 32: the application of CRISPR/Cas9 system in genome editing

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’

25

Page 33: the application of CRISPR/Cas9 system in genome editing

In term of cleavage :

- There is no different between cds or non-cds

locations and this is one of CRISPR/Cas9

advantages

26

Page 34: the application of CRISPR/Cas9 system in genome editing

third : what is our purpose to making a DSB in target DNA

27

Page 35: the application of CRISPR/Cas9 system in genome editing

Knock in

HDR :

homology

directed

repair

Knock out

NHEJ:

non

homologous

end joining

or

28

Page 36: the application of CRISPR/Cas9 system in genome editing

Gene of Interest

Protospacer Adjacent Motif (PAM)

Target Sequence

29

Page 37: the application of CRISPR/Cas9 system in genome editing

Non-Homologous End Joining (NHEJ)

and DNA repair pathway30

Page 38: the application of CRISPR/Cas9 system in genome editing

Transferring crRNA:tracrRNA-Cas9 complex to the cell nucleus

31

Page 39: the application of CRISPR/Cas9 system in genome editing

Non – viral based gene delivery :

- Lipid-Mediated Transfection

- Calcium phosphate transfection

- Electroporation

ghfgh

32

Page 40: the application of CRISPR/Cas9 system in genome editing

viral based gene delivery :

- Lentivirus

-Adenovirus

-Adeno-Associated Virus (AAV)

- crispr plasmid (pCRISPR)

33

Page 41: the application of CRISPR/Cas9 system in genome editing

34

Page 42: the application of CRISPR/Cas9 system in genome editing

35

Page 43: the application of CRISPR/Cas9 system in genome editing

Correction of Mutations in Zygote stages of Human?

We have more knowledge and techniques on Human Embryo than Monkey’s36

Page 44: the application of CRISPR/Cas9 system in genome editing

37

Page 45: the application of CRISPR/Cas9 system in genome editing

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

38

Page 46: the application of CRISPR/Cas9 system in genome editing

conclusion

Page 47: the application of CRISPR/Cas9 system in genome editing

Application of CRISPR/Cas9

- Knockout/Knockin Mouse generation :

Traditional methods: at least 6~12 monthsCRISPR/Cas9 : 2 Months with ~90% of efficiency

39

- 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

Page 48: the application of CRISPR/Cas9 system in genome editing

In Here, donor strand is a ssDNA

40