These are the part of the Bacterial immune system which detects and recognize the foreign DNA and cleaves it. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci Cas (CRISPR- associated) proteins can target and cleave invading DNA in a sequence – specific manner. CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes.

1. MBB – 607 – Advances In Genetic Engineering (3+0)
Topic - Recent advances in CRISPR-CAS9 technology: an alternative to
transgenic breeding
Department of Agricultural
Biotechnology
Presented By -
Jyoti Prakash Sahoo
01ABT/PHD/17
Dept. of Agril. Biotech.
OUAT, BBSR
Course Instructor –
Dr. I. C. Mohanty
Asst. Professor
Dept. of Agril. Biotech.
OUAT, BBSR

2. 2
Evolution of genome editing
techniques
 Genome editing, is a type of genetic engineering in which DNA is
inserted, deleted or replaced in the genome of a living organism
using engineered nucleases, or "molecular scissors.“
Homologous
Recombination
Site Specific
Recombination

3. CRISPR – Cas systems
• These are the part of the Bacterial immune system which detects and recognize the
foreign DNA and cleaves it.
1. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
loci
2. Cas (CRISPR- associated) proteins can target and cleave invading DNA in a
sequence – specific manner.
 CRISPR array is composed of a series of repeats interspaced by spacer sequences
acquired from invading genomes.
3
http://schaechter.asmblog.org/schaechter/2011/04/six-questions-about-
crisprs.html
o Array of alternating spacers and palindromic direct repeats.
o The identical repeats range between 21 and 47 bp in different loci
o Spacers are of constant length but are hypervariable in sequence
o CAS proteins that add new spacer-repeat pairs, process the CRISPR
transcript, and cleave the recognized foreign DNA.

4. 4
The Story of CRISPR – Cas9
Technology
first observed in Escherichia coli by
Osaka University researcher
Yoshizumi Ishino

5. 5
http://dharmacon.gelifesciences.c
om/
Key
components
http://takara.co.kr/file/manual/images/631439_01.jpg
Trans-active CRISPR
RNA

6. 6
Class 1- type I (CRISPR-Cas3) and type III (CRISPR- Cas10) - uses several
Cas proteins and the crRNA
Class 2- type II (CRISPR-Cas9) and type V (CRISPR- Cpf1) - employ a
large single-component Cas-9 protein in conjunction with crRNA and
tracerRNA.
Different CRISPR-Cas system in Bacterial Adaptive
Immunity
www.slideshare.net

7.  Cas9 protein has sixdomains
 The Rec I domain is the largest and is responsible for binding guide RNA.
 The role of the REC II domain is not yet well understood.
 The arginine-rich bridge helix is for initiating cleavage activity.
 The PAM-Interacting (Protospacer adjacent motif) domain responsible for
initiating binding to target DNA.
 The HNH and RuvC are nuclease domains that cut single-stranded DNA.
Structure of cas9 protein
7
M Jinek et al. 2014
http://sites.tufts.edu/crispr/crispr-mechanism/
(2 -6 bp)
25 alpha helix
and 2 bete
sheets

8.  The guide RNA is engineered to have a 5′ end that is complimentary
to the target DNA sequence.
 This artificial guide RNA bind Cas9, induces some conformational
changes, and make it active.
 Then both act on target DNA.
Structure of cr RNA
8
http://sites.tufts.edu/crispr/crispr-mechanism/

9. 9
http://sites.tufts.edu/crispr/crispr-mechanism/
Cas 9 Complex
(Activation)

10. 10
CRISPR – Cas 9 mediated immunity in bacteria
1. Cleavage
mechanism

11. 2. Repair mechanism
Non-
homologous
end joining
Homologo
us direct
repair
Knock Knock in
11
CRISPR – Cas 9 mediated immunity in bacteria

12. 12
Cpf1 is a single RNA-guided endonuclease of a class 2
CRISPR-Cas System – Alternate to Cas 9
 Two-component RNA-programmable DNA nuclease
 Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5′ T-rich
PAM
 Not required tracerRNA

13. 13
Possible experimental designs of CRISPR/Cas9
using wild-type
Cas9
using Cas9 nickase, leads subsequent NHEJ
response
using dCas9 and transcription activator
fusion with transcription activating proteins
(VP64)
using dCas9 reduces gene expression by steric
hindrance
using homology-directed repair
(HDR)
using by HDR from a single-stranded
oligonucleotides

14. General protocol for
CRISPR
14
www.slideshare.net

15. Plasmid delivery of
sgRNA/Cas9
15
 Plasmid construction is simple; design oligos specific to your target
sequence, and clone into the pre-linearized plasmid using the included
ligation reagents and high-efficiency competent cells.
http://www.calontech.com/

16. sgRNA designing
tools
 Optimized CRISPR Design (Feng Zhang's Lab at MIT/BROAD, USA)
 sgRNA Scorer (George Church's Lab at Harvard, USA)
 sgRNA Designer (BROAD Institute)
 ChopChop web tool (George Church's Lab at Harvard, USA)
 E-CRISP (Michael Boutros' lab at DKFZ, Germany)
 CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK)
16

17. General protocol for
CRISPR
www.slideshare.net
17

18. Strategy no 1:- Drive gRNA expression using a different
promoter
Stratergy no 2:- remove restrictions in the PAM
sequences
Stratergy no 3:- by editing the cas 9
sequence
1. Expanding CRISPR-CAS9 recognition
sequence
Recent Advances
18

19. 19
2. Improving cleavage specificity of the CRISPR-CAS9
system

20. 20
3. Inducible cas9
expression

21. 21
Some
applications
2. In an effort to confirm that gene editing was at least possible, cells from rice
plants
were transformed with vectors carrying CRISPR gateway vector targeting
CHLOROPHYLL A OXYGENASE 1 (CAO1) gene (Miao et al., 2013)
1. HepG2 cells expressing hepatitis B virus (HBV), the introduction of
CRISPR‐Cas9 system resulted in both decreased hepatitis B core antigen
expression which provides an impetus for further research on the possibility
of CRISPR‐Cas9‐mediated hepatitis B prevention

22.  Can be used to create high degree of genetic variability at precise
locus in the genome of the crop plants.
 Potential tool for multiplexed reverse and forward genetic study.
 Precise transgene integration at specific loci.
 Developing biotic and abiotic resistant traits in crop plants.
 Potential tool for developing virus resistant crop varieties.
 Can be used to eradicate unwanted species like herbicide
resistant weeds, insect pest.
 Potential tool for improving polyploid crops like potato and wheat.
Application in
Agriculture
22

23. 23
Reference
s

24. 24