1) The document discusses the CRISPR-Cas9 system of genome editing and its applications. 2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs. 3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.

1. PRESENTEDBY
K.A.SARAVANAN
M.V.Sc
DIVISION OF ANIMAL GENETICS
IVRI
GENOME EDITING WITH
CRISPR-Cas9

2. OUTLINE
Genome Editing
CRISPR – Cas
system of Immunity
History
Applications
New Developments
General Protocols
CRISPR-Cas9 in
genome editing
Comparisions with
ZFN & TALENS

3. GENOME EDITING
• Genome editing technique - 1990 - Kim et al.
• Nine genome editors were available as of 2017 (Church.G,2017)
DNA guided RNA guided Protein guided
TtAgo Group II Introns Meganucleases
λ-bet/exo MAGE CRISPR-cas9 Recombinases
Rec A CAGE ZFNs, TALENs

4. History of Genome editing
Source

5. Story of CRISPR/Cas9
Zhang et al,2014
F.MOJIKA

6. 2015 Breakthrough prize

7. Zinc Finger Nucleases (ZFNs)
• First Nuclease based technique for
genome editing (1998)
• Contains two domains
✓ Zinc Finger Peptide (ZFP) -
complementary sequence of
target DNA
✓ FokI restriction enzyme -
cleaves ds DNA where ZFP binds
with DNA
Source:

8. • Second technique commonly used
• Has two domains
✓Transcription activator-likeeffector domain
✓FokI restrictionenzyme
• Economical and manufacturing is easy as compared to ZFNs
Transcription Activator-Like Effector Nucleases
(TALENs)
Geibler R, et al. (2011)

9. CRISPR-Cas technique
• Advanced technique for genome editing
• Accurate method for gene modification
• Multiplexes gene editing
• Adaptive immune system of bacteria
CRISPR-Cas9
Clustered Regularly Interspaced Short Palindromic Repeats
Cas-CRISPR Associated protein
Palindromic repeats

11. ZFN vs TALENs vs CRISPR/Cas9
T.K.Guha et al.,2017
Target efficiency limiting average Good
Species few few Many
Dimerizationreq. yes yes No

12. CRISPR-Cassystemof Immunity
Has three stages
❑ Adaptation / recognition
❑ Expression
❑ Interference.
Found in
• 45% - bacterial genome
• 84% - Genome of archaea
Grissa et al,2007
Protospacers
PAM

13. IMMUNIZATION IMMUNITY
Rath D, et al.,2015

15. • Has two domains
✓ Guide RNA (sgRNA) - complementary to target sequence
✓ Cas9 enzyme - helicases and endonucleases
CRISPR-Cas 9 system
in genome editing
sgRNA
• CRISPR RNA (crRNA) and Trans-activating
crRNA (tracrRNA) connected by a short RNA
linker
• Specific RNA sequence that recognizesthe
target DNA region of interest and directs
the Cas nuclease there for editing

16. Cas
❖ CRISPR-associatedprotein - non-specific endonuclease
❖ Directed to the specific DNA locus by a gRNA, where it makes a double-strand
break
❖ Several versions of Cas nucleases isolatedfrom different bacteria
❖ Most commonly used one - Cas9 nuclease from Streptococcus pyogenes
Class 2
Types 16
Subtypes 33
Cas genes 93

17. Cas9 Structure and Working Mechanism
• Cas9 : Bi-lobed structure having active sites and two grooves for binding of
nucleic acid
- REC (large recognition lobe)
- NUC (small nuclease lobe)
• Specificity of Cas9 depends on REC
• NUC has two domains
1- RuvC & HNH
2- PAM interacting domain (PI).
Both are connected by a helix bridge
S. pyogenes Cas9

18. 1. PAM binding - locatingthe target sequence
- Initialbinding of Cas9 to PAM (N-G-G) sequences - quickly screen for
potentialtarget sequences
2. Phosphate Lock Loop Binding
- Stabilizes the target DNA
3. DNA recognition
-
DNA binding and cleavage

19. 4. Cleavage:
. - cleave very specifically between the 3rd & 4th nucleotides from the PAM.
❖HNH Nuclease - cleaves the DNA strand complementary to the RNA guide.
❖RuvC Nuclease - cleavage of the DNA strand not complementary to the
guide RNA

21. Repair process
Source: Sciencedirect

22. Ethan bier et al,2018

23. 1.Select genomic target
- 20 bp sequence followed by PAM (NGG)
- Use online toolsto minimize off-targeting
2.Design sgRNA
- guide sequence should match with target
3.Assemble cas9/sgRNA construct
4.Delivery
General protocol for CRISPR
TARGET SEQ
OFF TARGETS

25. Delivery methods
http://www.els.net [doi: 10.1002/9780470015902.a0026629]

26. Source:wikipedia

27. 1.Cas9 modifications
• dCas9 - inactive by producingpoint mutationin HNH & RuvC domains
• Lacks cleavage activity- cannot produce DSBs - used for the activation and
repression of specific gene (CRISPRa & CRISPRi)
• Cas9 Combines with Proteins (epigenetic factors , fluorescent proteins….)
NEW DEVELOPMENTS
Qi et al.,2013

28. ▪ Cas9 nucleases range from about 900 to
1,600 amino acids.
▪ Most commonly used - adaptedfrom the
type II-A CRISPR system from Streptococcus
pyogenes (Sp).
Species PAM Sequence
Streptococcus pyogenes (Sp) NGG
Staphylococcus aureus (Sa) NNGRRT(where R is an A or G)
Neisseria meningitidis NNNNGATT
S. thermophilus NNAGAAW, where W is an A
or T
2.Diversity of Cas9 Orthologs
Cebrian.S et al.,2017

29. • In 2015 - Chen et al.&
Zetche et al. - discovered a
new class of CRISPR
protein - Cpf1
(from Prevotella and
Francisella )
• 16 Cpf1 proteinsidentified
- only two are appropriate
for human.
• small in size
• easy to handle
Cpf 1
Zetsche et al.,2016

30. APPLICATIONS OF CRISPR-Cas9
out- NHEJ
Asad Razzaq et al.,2018

31. CRISPR in animals and animal models
Shrock et al.,2017
Bioreactors for pharmaceutical
production
Revive extinct species
Develop a universal source of donor
organs
Model for human diseases Use of CRISPR in farming

32. • Treat many genetic and viral diseases
• Cystic fibrosis treatment:First isolate intestinal stem cell & develop it in
culture in laboratory & then remove the targetallele (F508) & placed
back to the human.
GENE THERAPY
MarangiM et al,2018

33. SOURCE:

34. HIV

35. Fostemvir
Ibalizumab
HIV treatment using CRISPR
Ophinni et al,2018

36. CRISPR with Gene drive
Publications: Scopus; Patents: The Lens; Funding: NIH RePORTER.

37. Designer babies
Source: M. Araki & T. Ishii Reprod. Biol. Endocrinol. 12, 108 (2015)

39. SUMMARY

40. THANK
YOU
KAS
AGB