CRISPR-cas9 system is a new robust genome editing tool and also this is a really easy to use system. کاربرد سیستم کریسپر در ویرایش ژنوم.ppt

2. Introduction

3. CRISPR
C: Clustered
R: regularly
I: interspaced
S: short
P: palindromic
R: repeat

4. 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

5. 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

6. 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

7. 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

8. 5

13. 6

14. 7

15. Stage 1:
CRISPR spacer acquisition
8

16. Stage 2:
CRISPR expression
9

17. Stage 3:
interference
Interference system rely on :
-association of Cas protein with sgRNAs and making
ribonucleoprotein Complex (RNP)
10

18. Stage 3:
interference
11
RNP Complex

19. Stage 3:
interference
Interference system rely on :
- PAM: protospacer adjacent motifs
12

20. Stage 3:
interference
5'-NGG-3'
13

21. 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

22. 15

23. 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

24. - Cas-sgRNA complex is a smart and
programmable Nuclease
As stated before :
- RNP recognition sites is 17-20 nt
(spacer lenght)
17

25. 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

26. First : we have to designing spacer
19

27. - tracrRNA : trans-activating CRISPR RNA
- crRNA : CRISPR RNA
dsRNA
20

28. 21

29. 22

30. 23

31. second : Cas9 principles
24

32. 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

33. In term of cleavage :
-There is no different between cds or non-cds
locations and this is one of CRISPR/Cas9
advantages
26

34. third : what is our purpose to making
a DSB in target DNA
27

35. Knock in
HDR
:
homology
directed
repair
Knock out
NHEJ
:
non
homologous
end
joining
or
28

36. Gene of Interest
Protospacer
Adjacent Motif
(PAM) Target Sequence
29

37. Non-Homologous End Joining (NHEJ)
and DNA repair pathway
30

38. Transferring crRNA:tracrRNA-Cas9
complex to the cell nucleus
31

39. Non – viral based gene delivery :
-Lipid-Mediated Transfection
- Calcium phosphate transfection
- Electroporation
ghfgh
32

40. viral based gene delivery :
-Lentivirus
-Adenovirus
-Adeno-Associated Virus (AAV)
- crispr plasmid (pCRISPR)
33

41. 34

42. 35

43. Correction of Mutations in Zygote stages of Human?
We have more knowledge and techniques on Human Embryo than Monkey’s36

44. 37

45. pXK7-AtCas9-2
14488 bp
Egfp
complementary
comple 2
NLS-At
NLS-At
Cas9
attB1
attB2
Kan
Sm/SpR
p35S
T35S
LB
RB
3X FLAG
38

46. conclusion

47. Application of CRISPR/Cas9
- Knockout/Knockin Mouse generation :
Traditional methods: at least 6~12 months
CRISPR/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

48. In Here, donor strand is a ssDNA
40