Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR. Prepared in Oct 2015

1. Ashikh Seethy
Dept of Biochemistry, MAMC- New
Delhi
CRISPR/Cas9

2. Gene Editing:
 In vivo gene editing:
 Zinc Finger Nucleases (ZFN)
 TALENs
 Homing Endonucleases
(Meganucleases)
 CRISPR/Cas9

3. Introduction

5. Zinc Finger Nucleases:

6. Introduction

7. TALENS: Transcription Activator-Like
Effector Nucleases
 DNA binding domain: 33-34 amino acid sequence
•Secreted by Xanthomonas when they infect
various plant species
•Bind promoter sequences in the host plant
•Activate the expression of plant genes that
aid bacterial infection

8. Meganucleases or Homing
Endonucleases:
 5 families:
 LAGLIDADG
 GIY-YIG
 HN
 His-Cys box
 PD-(D/E)XK
 5 families:
 LAGLIDADG
 Recognizes 14-40 bp long sequences

9.  Protein based gene editing systems are difficult to
engineer
 Expensive
 Time consuming
 Off-targeting
 Alternative: CRISPR/Cas9 System

10. CRISPR: Clustered Regularly
Interspaced Short Palindromic Repeats
 1987

11. 1987
2000: CRISPR is present in > 90% archae and > 40%
bacteria
2002: Identification of cas- CRISPR Associated

12. 2005: Insights into Origin of Spacers

13. 2007: CRISPR Adaptive Immunity in
Bacteria

14. 2010: Cas9 Cleaves Target DNA via Double
Stranded Breaks

15.  crRNA: CRISPR RNA
 tracrRNA: trans-activating
CRISPR RNA
 PAM: Protospacer Adjacent
Motifs NGG in S.pyogenes
 sgRNA: single guide RNA aka
crRNA-tracrRNA chimera

16. Introduction

17. Introduction

18. Types of CRISPR/Cas Systems:

19. Designing CRISPR/Cas9:

20. Designing
CRISPR/Cas9:

21. Off Targeting by CRISPR/Cas9:

22. CRISPR Nickases: ↓Off-targeting
D10A
H840A

23. Gene manipulations with CRISPR/Cas9

24. Gene Regulation:
D10A
H840A

25. CRISPR/Cas9
Advantages
 Based on Watson-Crick base-pairing of sgRNA-DNA and an NGG PAM motif
straightforward and flexible
 Multiplex gene targeting
 Much larger targetable sequence space
Limitations
 The requirement of a protospacer adjacent motif (PAM) sequence limits the
number of potential target sequences
 Sequence specificity to target loci is only 14 nt long (12 nt of sgRNA and 2nt of the
PAM), which can recur around ~11 times in a human genome
 Endogenous chromatin states and modifications may prevent the sequence specific
binding

26. Introduction

27. Hereditary tyrosinemia Type I
 Fumaryl-acetoacetate hydrolase (FAH)
 Homozygous GA mutation in the last exon
 Liver failure and death in infancy

28. Introduction

30. Tool Against the World’s Deadliest Animal

32. Than
k