This document discusses genome editing techniques. It begins by defining genomes and how they consist of DNA or RNA that contains both coding and non-coding regions. It then discusses several methods of genome editing including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system. Each method uses engineered nucleases to introduce targeted double-strand breaks in DNA, allowing the cell's repair mechanisms to modify the genome. The CRISPR-Cas system was selected as the breakthrough of the year in 2015 due to its simplicity, efficiency and precision for genome editing applications.

2. Sudeep Pandey
PALB 6306
Department of
Plant Pathology

3. GENOME
• A genome is the genetic material of an organism. It consists of
DNA (or RNA in RNA viruses). The genome includes both
the genes (the coding regions), the noncoding DNA and the genetic
material of the mitochondria and chloroplasts.
• The term genome was created in 1920 by Hans Winkler, professor
of botany at the University of Hamburg, Germany.

4. GENOME EDITING
• the concept of genome editing is not limited to genes.
• also includes making alterations to non-coding regions of
genomes and to epigenomes.
• Targeted interventions
• Alter the structural or functional characteristics like
 colour or number of blooms in flowering plants
some disease traits in animals and plants

5. GENOME EDITING
• This approach is called reverse genetics
• Among the key requirements of reverse genetic analysis is the ability
to modify the DNA sequence of the target organisms.
• Genome editing was selected by Nature Methods as the 2011
Method of the Year. The CRISPR-Cas system was selected by as 2015
Breakthrough of the Year

6. Pathogens weapon Can be neutralized by
Pathogen produce virulence
factor
Suppression of Virulence Functions
Plants have susceptibility gene Genome editing
Many pathogens attack crop Introduction of novel R genes and stacking
multiple genes at a preferred site.
Pathogen has defeated many R
gene
Restore defeated R genes
Ways to produce disease resistance plant
(Sebastian, 2013)

7. Methods for
plant
genome
manipulation
Classical breeding
Transgenic approach
Targeted genome editing
An alternative to
both classical
plant breeding and
transgenic
approach

9. Comparison between traditional and modern genome editing
technologies
Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-
ray or fast neutron
radiation)
Biological (ZFNs, TALENs
or CRISPR/ Cas)
Biological- Transgenics
(e.g., Agro or gene gun)
Characteristics
of genetic
variation
Substitution and Deletion Deletion and
chromosomal mutation
Substitution and Deletion
and insertion
Insertions
Loss of function Loss of function Loss of function and gain
of function
Loss of function and gain
of function
Advantages Not necessary of knowing gene
function or sequences
Not necessary of knowing
gene function or
sequences
Gene specific mutation Insertion of genes of
known functions into host
plant genome
Easy production of random
mutation
Easy production of
random mutation
Efficient production of
desirable mutation
Efficient creation of plants
with desirable traits
9

10. Disadvantages Inefficient screening of
desirable traits
Inefficient screening
of desirable traits
Necessity of knowing
gene function and
sequences
Necessity of knowing
gene function and
sequences
Non specific mutation Non specific
mutation
Prerequisite of
efficient genetic
transformation
Prerequisite of
efficient genetic
transformation
Other features Non transgenic process
and traits
Non transgenic
process and traits
Transgenic process
but non transgenic
traits
Transgenic process
and traits
10
Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-
ray or fast neutron
radiation)
Biological (ZFNs, TALENs
or CRISPR/ Cas)
Biological- Transgenics
(e.g., Agro or gene gun)

11. Introduction…
• Genome editing, or genome editing with engineered
nucleases (GEEN) is a type of genetic engineering in which DNA is
inserted, replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
• The nucleases create specific double-strand breaks (DSBs) at desired
locations in the genome and harness the cell’s endogenous
mechanisms to repair the induced break by natural processes
of homologous recombination (HR) and non-homologous end-
joining (NHEJ).
11

12. Non-homologous end-joining (NHEJ) Homologous recombination (HR)
Rejoins the broken ends and is often
accompanied by loss/gain of some
nucleotides
Repair DNA as a template to restore the DSBs
Thus the outcome of NHEJ is variable Outcome of this kind of repair is precise and
controllable
(Hyongbum, 2014)

13. Why genome editing?
 To understand the function of a gene or a protein, one interferes with it
in a sequence-specific way and monitors its effects on the organism.
 In some organisms, it is difficult or impossible to perform site-specific
mutagenesis, and therefore more indirect methods must be used, such as
silencing the gene of interest by short RNA interference (siRNA).
But sometime gene disruption by siRNA can be variable or incomplete.
Nucleases such as CRISPR can cut any targeted position in the genome and
introduce a modification of the endogenous sequences for genes that are
impossible to specifically target using conventional RNAi.
13

14. Requirement :
A homing device: for specific identification of target sequence
An endonuclease: for creating double strand break
Uses:
 Gene knock out
 Gene tagging
 Specific mutation (insertion/deletion study)
 Gene knock in
 Promoter study
5

15. Novel
tools for
genome
editing
Zinc finger
nuclease
(ZFN)
TALENs
CRISPER/Cas9
Meganuclease
6
Transcription activator like effector nucleases
Clustered Regularly Interspersed Short
Palindromic Repeats (CRISPR)- CRISPR
associated (Cas) protein

16. 1. Mega Nuclease
 First tool used for double strand break-induced genome manipulation
 Occur naturally in Yeast and in Chlamydomonas
 In these enzymes binding site and restriction site occur within same unit
hence difficult to modify
 Crop where it is used
Crop/plant Trait Reference
Maize Herbicide resistance Gao et al, 2010
Cotton Herbicide resistance
Insect resistance
D’Halluin et al., 2013
Limitation
-Difficult to manipulate the DNA binding site
-Small recognition site

17. 2. Zinc finger nuclease
Zinc finger protein
 They were first identified as a DNA-binding motif in
Transcription factor TFIIIA from African clawed frog
(Xenopus laevis)
 Small protein structural motif that is characterized by
the coordination of one or more zinc ions in order to stabilize the
fold
 contain multiple finger-like protrusions that make tandem
contacts with their target molecule
These are hybrid restriction enzymes
Zn
H
HC
C
Consist of two parts: generated by fusing a zinc finger DNA-binding
domain to a DNA-cleavage domain

18. FokI, naturally found in Flavobacterium okeanokoites
N-terminal binding domain and a non-
specific DNA cleavage domain at the C-
terminal
Fok1

19. DEVELOPMENT OF ZINC FINGER NUCLEASE
 Zinc finger domains can be engineered to target specific
desired DNA sequences and this enables zinc-finger nucleases
to target unique sequences within complex genomes.
 By taking advantage of endogenous DNA repair machinery,
these reagents can be used to precisely alter the genomes of
higher organisms.

20. Mode of action
1.Binding of ZFN to DNA
2.Restricting the DNA
3.Cut sequence may be deleted/new
sequence may be added
4.Break end will
be sealed by host
own repairing
mechanism
5’ 3’
3’ 5’
+
-

21. Crop where it was used
Crop/plant Trait Rererence
Maize Herbicide tolerance Shukla et al., 2009
Soybean Physiological trait Curtin et al., 2011
Tomato against TYLCV Takenaka et al., 2007
12
Limitation
 Off target effect
 Construction is cumbersome and time consuming

22. 3. Transcription activator like effector nucleases (TALENs)
First time reported by Ulla Bonas in Xanthomonas
oryzae (1989)
Bacterial cell
Consist of TALE + Endonuclease
Prof. Ulla Bonas
Plant cell
Nucleus
Effector
Divert metabolic
machinery of host
towards the
pathogen
15

23. TAL (transcription activator-like) effectors are proteins
secreted by Xanthomonas bacteria via their type III secretion
system when they infect various plant species. These proteins
can bind promoter sequences in the host plant and activate
the expression of plant genes that aid bacterial infection.
 Transcription activator-like effector nucleases (TALEN)
are restriction enzymes that can be engineered to cut specific
sequences of DNA.
 They are made by fusing a TAL effector DNA-binding domain to a
DNA cleavage domain (a nuclease which cuts DNA strands).
Transcription activator-like effectors (TALEs) can be engineered
to bind practically any desired DNA sequence, so when
combined with a nuclease, DNA can be cut at specific locations.
16

24. An N-terminal domain
containing a type III
secretion signal
A central repeat domain
that determines DNA-
binding specificity
TALEs are organized into three sections
a C-terminal domain
containing a nuclear
localization signal and an
acidic activation domain
A stretch of 34 amino acid repeated at 15.5 - 19.5 times
……… ………
34 amino acid
12
Repeat variable diresidues (RVD)
13
In each repeat amino acid at the position 12 and 13 varies thus form a Repeat
variable diresidues(RVDs)
The amino acid identity of the RVDs is responsible for DNA nucleotide recognition,
enabling the design of TALENs to target unique DNA sequences
Molecular structure of effector

25. Once a DNA target is identified, an RVD for each target base is selected
according to the following code:
Designing TALEN
G NN, NH, NK
DNA base Amino acid in TALE
C HD
T NG
A NI
Bio-informatic tools available for predicting Binding site
Programme Website Refernece
Target Finder (https://tale-nt.cac.cornell.edu/) Doyle et al., 2012
Talvez (http://bioinfo.mpl.ird.fr/cgibin/talvez/talvez.cgi) Pérez-Quintero et al., 2013
Storyteller (http://bioinfoprod.mpl.ird.fr/xantho/tales) Pérez-Quintero et al., 2013
(Mussolino and Cathomen, 2012)

26. Mode of action
Fok1
Fok1
Fok1
5’ 3’
3’ 5’
Fok1
+
_
1. Binding of TALEN
2. Cutting at
target site
3 In/del
5’ 3’
3’ 5’
4. Gap sealing

27. Different ways by TALENs can be use for Disease resistance plant
• Transcription activation of different R gene
• Transcription Repression of different Susceptibility Gene
• Mutation in Promoters of Susceptibility Genes
• Gene replacement
• Destroying pathogen genome
(Sebastian,2013)

28. Examples of targeted gene modification using TALENs in plants
(Yong Zhang, 2013

29. 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.
A CRISPR array is composed of a series of repeats interspaced by spacer
sequences acquired from invading genomes.
29

30. 1987
• Researchers find CRISPR sequences in Escherichia coli, but do not
characterize their function.
2000
• CRISPR sequence are found to be common in other microbes.
2002
• Coined CRISPR name, defined signature Cas genes
2007
• First experimental evidence for CRISPR adaptive immunity
2013
• First demonstration of Cas9 genome engineering in eukaryotic cell
HISTORY

31. Components of CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)

32. Different Cas proteins and their function
Protein Distribution Process Function
Cas1 Universal Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types
Cas2 Universal Spacer acquisition specific to U-rich regions; present in all Types
Cas3 Type I signature Target interference DNA helicase, endonuclease
Cas4 Type I, II Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB
Cas5 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas6 Type I, III crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas7 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas8 Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of
CASCADE
Cas9 Type II signature Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like
nuclease domain; necessary for interference and target cleavage
Cas10 Type III signature crRNA expression
and interference
HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE
elements 32

33.  Protospacer adjacent motif (PAM) is a DNA sequence
immediately following the DNA sequence targeted by
the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
 PAM is a component of the invading virus or plasmid, but is not a
component of the bacterial CRISPR locus. Cas9 will not successfully
bind to or cleave the target DNA sequence if it is not followed by the
PAM sequence.
 PAM is an essential targeting component (not found in bacteria)
which distinguishes bacterial self from non-self DNA, thereby
preventing the CRISPR locus from being targeted and destroyed by
nuclease

34. 34
Structure of crRNA

35. Trans-activating crRNA (Tracr) RNA
A trans-encoded small RNA with 24 nucleotide complementarity to the repeat
regions of crRNA precursor transcripts
Function
 Pair with Cr RNA for its maturation by processing through RNAseIII
 Activating Cr RNA-guided cleavage by cas 9
(Jinek, 2012)

36. Action of CRISPR in bacteria
The CRISPR immune system works to protect bacteria from repeated
viral attack via three basic steps:
(1) Adaptation
(2) Production of cr RNA
(3) Targeting
36

37. 37

38. Versatile Nature of CRISPR Technology
38Jeffry et al., 2014

39. On line designing tools
Software Work
ZiFit
(http://zifit.partners.org/ZiFiT/)
Helps to construct gRNAs,
TALENs, and ZFNs targeting the
sequence of interest
CRISPR designing tools
(http://crispr.mit.edu/)
Helps design gRNA sequences
that are predicted to minimize
off-target mutations
E-CRISP (http://e-crisp-
test.dkfz.de/E-CRISP/
index.html)
Permits the finding of paired
gRNAs and off targets
CRISPR-PLANT Database
(http://www.genome.arizona.edu/
crispr/index.html)
An online tool that includes more
plant genomes
On line discussion group
(https://groups.google.com/forum/#!forum/talengi- neering;
(https://groups.google.com/forum/#!forum/crispr).
(Jorge Lozano, 2014)27

40. 40
General protocol for CRISPR

41. 41

42. What makes CRISPR system the ideal genome engineering technology
42

43. Examples of crops modified with CRISPR technology
43
CROPS DESCRIPTION REFERNCES
Corn Targeted mutagenesis Liang et al. 2014
Rice Targeted mutagenesis Belhaj et al. 2013
Sorghum Targeted gene modification Jiang et al. 2013b
Sweet orange Targeted genome editing Jia and Wang 2014
Tobacco Targeted mutagenesis Belhaj et al. 2013
Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpeng et
al. 2014
Potato
Soybean
Targeted mutagenesis
Gene editing
Shaohui et al., 2015
Yupeng et al., 2015
Harrison et al., 2014

44. Application in Agriculture
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.
44

45. Some pitfalls of this technology
Proper selection of gRNA
Use dCas9 version of Cas9 protein
Make sure that there is no mismatch within
the seed sequences(first 12 nt adjacent to
PAM)
Use smaller gRNA of 17 nt instead of 20 nt
Sequence the organism first you want to
work with
Use NHEJ inhibitor in order to boost up
HDR 45
Solutions
Off target indels
Limited choice of PAM sequences

46. How to avoid off-target effects?
- Optimization of Injection conditions (less cas9/sgRNA)
- Bioinformatics : Find a sgRNA target for less off-targets
“CRISPR Design” (http://crispr.mit.edu)
46

47. Final Conclusion

Crop improvement requires the constant creation and use of new
allelic variants
Genetic modification, including plant breeding, has been widely
used to improve crop yield and quality, as well as to increase
disease resistance
Progress in site specific nuclease coupled with increase crop
genome sequencing and more effective transformation system
offers great promise in creating non transgenic crops with
predetermined trait
TALENs, CRISPR/Cas, and ZFN can be easily fashioned to bind
any specific sequence of DNA (TALEs, CRISPR/Cas) because of
the simple rules governing their interactions with nucleic acids.

48.  Using these technologies ( ZFNs, TALENs, and
CRISPR/Cas9 ) plant genome can be successfully modified
Among the different genome editing tools after TALEN,
CRISPER/Cas9 is getting more popularity owing to specticity
simplicity ease in construction
A step ahead from the fear of transgenic.
Final Conclusion