Advanced genome & epigenome editing tools For manupulation of useful microbes/strains& their applications

1. BY
Assist. Prof.
Dr. Berciyal Golda. P
VICAS
Advanced genome & epigenome editing tools
For manupulation of useful microbes/strains&
their applications

2. Introduction
Genome is a fancy word for all your DNA. From potatoes
to puppies, all living organisms have their own genome. Each
genome contains the information needed to build and maintain
that organism throughout its life.
an organism, including a virus. The genome
is the full complement genetic information in a cell,
and contains the programme required for that
cell to function.

3. WhatisAGENOME?
• 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.
• The genome broadly refers to the total amount of DNA of a single
cell (haploid cell in the case of a diploid organism) of an
organism, including its genes.
“The whole hereditary information of an organism that is
encoded in the DNA”

4. Genes provide the information for making all proteins that are
necessary for the expression of characters.
Characters refers to how an organism looks, its physiology, its
ability to fight infections and even its behavior.
GENE PROTEIN CHARACTER
The genome is found inside every cell, and in those that have
nucleus, the genome is situated inside the nucleus. It is a part of the DNA
molecule.
DNA sequencing techniques enables scientists to determine the exact
order or sequence of the bases of a genome.

6. • The sequence information of the genome will show,
• the position of every gene along the chromosome,
• the regulatory regions that flank each gene &
• the coding sequence that determines the protein produce by each
gene.
WHY SEQUENCE GENOMES?
• Because there is a need to put information about the genomes of flora and
fauna in the context of the fields that they serve.
• Genomic sciences will serve those that choose genetic modification as a
method for crop improvement as well as those that apply conventional
breeding methods to improve and develop agricultural practices.

7. • This information is used by physiologists and scientists in research
determining relationships between stress, genes and yield potential etc.
• It can also be used to produce sufficient amounts of safe and nutritious
food in times of increased population growth.
• It can be used to conserve and protect agricultural and other environments.
• It can serve the farmer/producer under increasing financial pressure by
providing higher yields through improve varieties.
• It can be used to conserve and protect agricultural and other environments.

8. • We need information and technology to
– improve human health,
– harness natural energy,
– understand and react in a positive manner to global climate change,
– clean up our environment and
– ensure food safety.

9. WHAT IS GENOMICS?
• Genomics is the sub discipline of genetics devoted to the
–mapping,
–sequencing ,
–and functional analysis of genomics.
The field includes studies of introgenomic phenomena such as
heterosis, epistasis, pleiotropy and other interactions between loci
and alleles within the genome.

10. How is Genomics different from Genetics?
– Genetics looks at single genes, one at a time, like a picture
or snapshot.
-Genomics looks at the big picture and examines all the
genes as an entire system.

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

12. • GENOMEEDITING :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

13. 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)

14. Methods for
plant
genome
manipulation
Classical breeding
Transgenicapproach
Targetedgenome editing
An alternative to
both classical
plant breeding and
transgenic
approach

15. 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)
Characteristic
s 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

16. 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)
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
transformatio
n
Prerequisite of
efficient
genetic
transformatio
n
Other features Non transgenic
process and traits
Non transgenic
process and
traits
Transgenic
process but non
transgenic traits
Transgenic
process and
traits
10

17. GENOMEEDITING
• 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
of homologous recombination (HR)
mechanisms to repair the induced break by natural processes
and non-homologous end-
joining (NHEJ).
11

18. 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)

19. 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

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

21. 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

22. 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

23. 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
H
C
C
Consist of two parts: generated by fusing a zinc finger DNA-binding
domain to a DNA-cleavage domain

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

25. 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.

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

27. 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

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

29.  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.
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.

30. 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
containing a
domain
nuclear
localization signal and an
acidic activation domain
Astretch of 34 amino acid repeated at 15.5 - 19.5 times
…
Repeat variable diresidues (RVD)
12 13
……… ……
34 amino acid
The amino acid identity of the RVDs is responsible for DNA nucleotide recognition,
enabling the design of TALENs to target unique DNAsequences
In each repeat amino acid at the position 12 and 13 varies thus form a Repeat
variable diresidues(RVDs)
Molecular structure of effector

31. Once a DNA target is identified, an RVD for each target base is selected
according to the following code:
Designing TALEN
T
G
NG
NN, NH, NK
DNAbase
A
Amino acid in TALE
NI
C HD
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)

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

33. 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

34. Examples of targeted gene modification using TALENs in plants

35. CRISPR–Cassystems
• 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

36. 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

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

38. 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

39.  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

40. 34
Structure of crRNA

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

42. 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

43. 37

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

45. 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/
An online tool that includes more
plant genomes
crispr/index.html) On line discussion group
(https://groups.google.com/forum/#!forum/talengi- neering;
(https://groups.google.com/forum/#!forum/crispr).
27

46. 40
General protocol for CRISPR

47. 41

48. What makes CRISPR system the ideal genome engineering technology

49. 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

50. 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

51. 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 gRNAof 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

52. 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

53. 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.

54.  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