Various genome editing techniques available, their mechanism and importance in crop improvement

1. GENOME EDITING
Prepared by,
ADITHYA P BALAKRISHNAN
Reg. No: 04-AGRMA-01724-18
M. Sc. (Agri.)
Dept. of Genetics and Plant Breeding
C. P. C. A, S. D. A. U
Submitted to,
Dr. KAPIL TIWARI
Assistant Professor
Dept of GPB
S. D. A. U

2. CENTRAL DOGMA OF LIFE
DNA
RNA
PROTEIN
NATURAL DNA REPAIR MECHANISMS
Direct reversal
• Photoreactivation & Alkylation
ss Repair
• Excision repair & Mismatch
repair
ds Repair
• HR, NHEJ, Microhomology
mediated joining
Transcription
Translation

3. WHAT IS GENOME EDITING ?
 The way of making specific changes to DNA of a cell or an organism
 Also known as,
Gene editing/ Genome engineering
 Results in: Altered gene expression and protein behavior

4. KNOCKOUT KNOCKDOWN & KNOCKIN
GENE TARGETING

5. SOME HISTORICALASPECTS
Hussain, 2018

6. AVAILABLE TECHNIQUES
GENOME EDITING
TECHNIQUES
Conventional
genome
editing systems
Chemical
system
Protein based
nuclease
system
Homing
endonuclease
system
RNA-protein
based system
Techniques
in research
Conventional
homologous
recombination
ssODNs
homologous
recombination
Peptide NA
systems
Meganuclease
system
ZFN systems
TALEN systems
Adeno-
associated
virus (AAA)
systems
CRISPER
system
Type-1
CRISPER-Cas
Type-2
CRISPER-Cas
Type-3
CRISPER-Cas
ODN
techniques
Using novel
proteins
-Argonautes,
Integrases,
Recombinases
Synthetic
genomicsKhan, 2019

7. GENOME EDITING BY USING
CHIMERIC ENZYMES
DNA
Binding
Domain
Endonu
cleases
Domain
CHIMERIC
ENZYME/
DESIGNER
ENGINEERED
ENDONUCLEA
SES

8. BASIC STRUCTURE AND FUNCTION
OF ENGINEERED NUCLEASES
Double stranded breaks
Repair DSBs

9. MECHANISMS FOR REPAIR OF DSBs
Non Homologous End Joining
(NHEJ)
Two DNA ends ligated without
homology
Error-prone process
Chances of ‘indels’ (FS Mutations)
Predominates in the absence of
externally added homologous DNA
Homologous Recombination
(HR)
Resolves DSBs by replacing the
genetic material between two areas of
homology
 Introduce exogenous
sequences/alternate alleles

10. DSB resolution via HR or NHEJ
Dunn et al. 2014

11. Genome
Editing
With
Engineered
Nucleases
(GEEN)

12. MEGANUCLEASES
Microbial
endodeoxyribonucleases with
long recognition sequences (20-
40bp)
Discovery: late 1980s
Most specific naturally
occurring restriction enzymes
Two main enzyme families
Eg; I-CreI, I-SceI
Meganucleases
Intron
endonucleases
Intein
endonucleases

13. Zinc Finger Nucleases System
Artificial endonuclease system
Consists of two domains:
DNA binding domain Endonucleases domain
• Zinc Finger Motif
• TF III of Xenopus leavis
• one ZF domain binds to three
nucleotides
• Non-specific type II restriction enzyme
• FokI (Flavobacterium okeanokoites)
• Cleave on dimerization (So, add half to one
ZFNs)
Dunn et al. 2014

14. Step by Step Zinc-Finger Nuclease-Induced Genome Editing
Khan, 2019

15. Methods for Construction
OPEN
 Oligomerized pool engineering
Creating a pool of ZFAs, then
assessing them for use
Less efficient & Labour
intensive
CoDA
Context-dependent assembly
Individual ZFs were tested to
assess their target specificity
when placed in combination with
other ZFs

16. Transcription Activator-like Effector
Nucleases
Site specific artificial nucleases
Two domains:
DNA binding domain
Endonucleases domain• Transcription Activator-like Effectors
(TALEs)
• Xanthomonas
• 34 conserved proteins & repeat-
variable di residues (RVDs)
• One domain specifically bind to one nt
• Non-specific type II restriction enzyme
• FokI (Flavobacterium okeanokoites)
Dunn et al. 2014

17. Diagram Showing Mechanisms of Transcription Activator-like Effector Nucleases
Khan, 2019

18. CRISPR System
CLUSTERED
REGULARLY
INTERSPACED
SHORT
PALIANDROMIC
REPEATS
Cas 9: CRISPR associated
protein
Prokaryotic immune response
system extended as a genome
editing tool
C
R
I
S
P
R

19. Adaptive Immune System of Bacteria

20. CRISPR/Cas9 System
Guide RNA = crisprRNA + trans-activating crRNA
PAM = Protospacer adjacent motif

21. Schematic Demonstrating the Concept of CRISPR/Cas9 Interactions Leading to the
Destruction of Viral Genome at the Selected Splice Site by the crRNA/gRNA
Khan, 2019

22. COMPARISON
Khan, 2019

23. GENOME EDITING FOR CROP
IMPROVEMENT
Chen. 2019

24. Gene editing for precision plant breeding
Sanskriti et al. 2019

25. PROS and CONS
Safety risk
Ethical issues
Bioweapons
Reduced diversity
Immunotherapy
Cure genetic diseases
Drug development
Improve crops

26. REFERENCES
Dunn, D. A and Pinkert, C. A. (2014). Gene Targeting
Khan, S. H. (2019). Genome editing technologies: concept, pros and cons various
genome editing technologies and bioethical concerns for clinical applications
Jennifer A. Doudna and Emmanuelle Charpentier (2014). The newfrontier
ofgenome engineering with CRISPR-Cas9