Progress and prospects in plant genome editing

1. 16-April-18 1PG seminar
Welcome
Anilkumar, C.
PALB 5062
PhD scholar

2. Seminar
on
Progress and prospects in plant
genome editing
16-April-18 2PG seminar

3. Introduction
Novel tools for genome
editing
Practical applications
Case studies
In this session----
16-April-18 3PG seminar

4. 16-April-18 PG seminar 4
Introduction
Forward genetics phenotype to genotype
Reverse genetics genotype to phenotype
“Mutate genes then examine phenotypes”
Strategy: Systematically inhibit the function of
every gene in a genome
• Approach 1: gene trap mutagenesis / Insertional
Mutagenesis
• Approach 2: inhibit gene expression using RNA
interference measure the effect of gene
disruption on a phenotype
Reverse genetics

5. 16-April-18 PG seminar 5
Methods for plant genome manipulation
Classical Breeding Transgenic Approach
Targeted Genome Editing
An alternative to both classical plant breeding and transgenic approach

6. 16-April-18 PG seminar 6
Principle
DNA repair system works when their will be DNA double strand breaks (DSBs)
Non-homologous end-joining (NHEJ)
 Rejoins the broken ends and is often
accompanied by loss/gain of some
nucleotides
 Thus the outcome of NHEJ is
variable
Genome Editing
A type of genetic engineering in which DNA is inserted, replaced, or removed
from a genome using artificially engineered nucleases, or molecular scissors.
Homologous recombination (HR)
 Outcome of this kind of repair is
precise and controllable
 Repair DNA as a template to restore
the DSBs
(Hyongbum, 2014)

7. 16-April-18 PG seminar 7
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

8. 16-April-18 PG seminar 8
Novel tools for Genome Editing
Zinc
finger
nuclease
(ZFN)
CRISPR/Cas9
Meganuclease
TALENs- Transcription activator like effector nucleases
CRISPR- Clustered Regularly Interspersed Short Palindromic Repeats
CAS Protein - CRISPR associated Cas 9 protein
TALENs

9. 16-April-18 PG seminar 9
A brief history of genome editing tools
Zinc Finger technology was presented by Pavletich and Pabo in 1991
in the journal Science.
 In 2009 the genome targeting abilities of TAL effectors was
published and was used for genome editing and thus TALEN, emerged..
 Potential target sites and simple method of building TAL effector
arrays, it was named “Method of the Year 2011” by journal Nature.
In 2012 CRISPR was demonstrated as a new genome editing tool.
Identified in case of bacteria Streptococcus pyogenes.

10. 16-April-18 PG seminar 10
1. Mega nuclease
 First tool used for double strand break-induced genome manipulation
 Occur naturally eg. I-Scal in Yeast and I- CreI 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 (Hyongbum, 2014)

11. 16-April-18 PG seminar 11
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:
Fig: Restriction endonuclease FokI, C terminal
Fok1
FokI, naturally found in Flavobacterium okeanokoites
Type IIS (asymmetric recognition site with cleavage occurring at defined region)
restriction enzyme
N-terminal binding domain and a non-specific DNA cleavage domain at the C-
terminal

12. 16-April-18 PG seminar 12
How to design a zinc finger nuclease
Choose a DNA segment of
interest and designing the
coding sequence for zinc
finger protein binding to it
These coding sequences are linked to that of the nonspecific
cleavage domain from the FokI restriction endonuclease
with the help of spacer add nuclear localization signal
Take Nonspecific cleavage
domain from the FokI restriction
endonuclease
Clone fused sequence
in expression vector
Purify the protein
Test in vitro activity
Clone in binary vector
Agromobilzation
Plant transformation

13. 16-April-18 PG seminar 13
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’
+
-
16-April-18 PG seminar 13
Crop where it was used
Crop/plant Trait Reference
Maize Herbicide tolerance Shukla et al. (2009)
Soybean Physiological trait Curtin et al. (2011)
Tomato against TYLCV Takenaka et al. (2007)
Limitation
 Off target effect
 Negative impact on cell proliferation
 Construction is cumbersome and time consuming

14. 3. Transcription activator like effector nucleases (TALENs)
First time reported by Ulla Bonas in Xanthomonas
oryzae (2011)
Bacterial cell
Consist of TALE + Endonuclease
Prof. Ulla Bonas
Plant cell
Nucleus
Effector
Divert metabolic
machinery of host
towards the
pathogen
TTSS

15. 16-April-18 PG seminar 15
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

16. 16-April-18 PG seminar 16
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

17. 16-April-18 PG seminar 17
Different ways by TALENs can be use for Disease
resistance plant
Transcription activation of different R gene
Mutation in Promoters of Susceptibility Genes
Transcription Repression of different Susceptibility Gene
Destroying pathogen genome
Gene replacement
(Sebastian, 2015)
Examples of targeted gene modification using TALENs in plants
(Yong Zhang, 2014)

18. 16-April-18 PG seminar 18
4. Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-
CRISPR associated (Cas) protein
Consist of two parts
Repeats of 20-50bp are
derived from the foreign DNA
Act as the memory to
the invading
pathogen
Produce cri RNA Tracr RNA+
crRNA:tracrRNA hybrids
Cri RNA
Tracr RNA
1CRISPR 2 Cas
1. CRISPR
A CRISPR locus is defined as an array of short direct repeat interspersed with
spacer sequences
Promoter SpacerRepeat Repeat RepeatSpacer
CRISPR Locus

19. 16-April-18 PG seminar 19
Promotor Repeat RepeatSpacer Repeat Spacer Spacer
Transcription
Spacer
Bacterial cell
Genesis of new Repeat region
Cleaved by cas nuclease
repeat
CRISPR locus

20. 16-April-18 PG seminar 20
Cr
Mode of action of CRISPR/Cas system
crRNA:tracrRNA hybrids
Protospacer region
CRISPR
tracr RNA
Transcription of a CRISPR
repeat-spacer array
Biogenesis of crRNA and trRNA
Cas
Cas9:crRNAtracr
RNAcomplex
Cr RNA
Cas Cr RNA

21. 16-April-18 PG seminar 21
Genome editing tools in nutshell
Property ZFN TALEN CRISPR
Binding Principle Protein-DNA Protein-DNA RNA-DNA
Core component ZFP-Fok1 fusion TALE-Folk1
Fusion
Sg RNA and
Cas9
Design Moderate Easy Very easy
Construction 5-7 days 5-7 days 1-3 days
Cost high moderate low
Efficiency Variable high high
Off target high low high
Length of target
sequence
18-24 bp
including spacer
50-60bp
including spacer
⁓20 bp

22. 16-April-18 PG seminar 22
Practical applications
In functional genomics:
• These include creation of point mutations, insertion of new genes
in specific locations or deletion of large regions of the nucleotide
sequences, and correction or substitution of individual genetic
elements and gene fragments.
• Selective binding of SSNs help to regulate gene action.
• These approaches also helps to identify genes involved in crop
domestication.
In crop improvement:
• To insert point mutations similar to natural SNPs
• To make small modifications to gene function
• For integration of foreign genes
• For gene pyramiding and knockout
• For the repression or activation of gene expression
• Modifying susceptibility genes (S-genes) and resistance genes (R-
genes),

23. 16-April-18 PG seminar 23
Case study:1
Objectives of the study:
• To Edit a specific Susceptibility gene(Os11N3) in rice to affect the
virulence strategy of Xanthomonas oryzae pv. oryzae.
• To Engineer heritable genome modifications to devastating
Bacterial blight of rice

24. 16-April-18 PG seminar 24
Nucleus
Leaf Cell
TAL effector
Os11N3
Glucose
Xanthomonas
TAL effector
Os11N3
EBE for AvrXa7
EBE for PthXo3
(Li et al., 2012)
(EBE- effector
binding element)

25. 16-April-18 PG seminar 25
Designing of TALENs targets promoter region of Os11N3 gene
dTALE-L1 & dTALE-R1 : designed
TALEs for PthXo3
AvrXa7 : Native TALEs
dTALE-R2 : designed TALEs for
AvrXa7
Black underlined : EBE for AvrXa7
Red underlined : EBE for PthXo3
Boxed : TATA box
(Li et al., 2012)
• Deployment of 2 pairs of TALENs to induce mutations in the overlapping EBEs of Os11N3 promoter.
• This helps in interfering with the virulence function of AvrXa7 and PthXo3 but not the develpmental
function of Os11N3

26. 16-April-18 PG seminar 26
Transforming the construct into Kitake rice embryonic cells using
Agrobacterium tumefactions
Analysis of the selected transformants to detect potential sequence
alterations
Performance of bacterial infection assays using leaf-tip clipping
method
(Li et al., 2012)
Procedure

27. 16-April-18 PG seminar 27
Disease resistance in rice T1 plants
Lane 1-20 : T1 mutant plants
Lane 21-22 : Wild type Kitake plant
Resistance : Lesion length of 1-4 cm
Susceptible : Lesion length of 10-14 cm
(Li et al., 2012)

28. 16-April-18 PG seminar 28
Case study:2
Objectives of the study:
• To Edit three MILDEW RESITANCE LOCUS (MLO) in wheat to provide
broad spectrum resistance against powdery mildew
• To Find the feasibility of the targeted foreign DNA insertion in bread
wheat through non homologous end joining

29. 16-April-18 PG seminar 29
Wheat protoplast transformation
Biolistic transformation of wheat
Protoplast transformation was carried out with 20 μg of TALEN plasmid per
transformation, or a mixture of 10 μg pJIT163-Ubi-Cas9 plasmid and 10 μg
pU6-gRNA plasmid
Screening of site specific nuclease induced mutation
Powdery mildew infection and microscopic analysis
(Wang et al., 2014)

30. 16-April-18 PG seminar 30
Analysis of genomic DNA of wheat transformed protoplast using
PCR restriction assay
Lanes “1”, digested T-MLO-transformed
protoplasts
L=Lanes “2” and “3”, digested and undigested
wild type
Red arrowheads, bands with mutations
Outcome of PCR/RE assay
Sequences of T-MLO-induced mutations in
the three MLO homoeoalleles in the
protoplasts
The numbers at the side indicate the type of
mutation and how many nucleotides are
involved
(Wang et al., 2014)

31. 16-April-18 PG seminar 31
Impact on disease resistance
Loss of TaMLO function confers resistance of bread wheat to powdery mildew disease
All of the combinations of the TaMLO-A1, TaMLO-B1 and TaMLO-D1 homozygous
mutants (tamlo-aa, tamlo-bb, tamlo-dd, tamlo-aabb, tamlo-aadd and tamlo-aabbdd) were
obtained by selfing
Seedling leaves of these mutants were inoculated with the virulent race of B. graminis f.sp.tritici
No microcolony
Micograph of microcolony foramtion on leaf surface after 3 d post inoclation
Macroscopic infection phenotype of representative leaves after 7 days post inoculationDisease symptoms of wild-type (WT) and tamlo-aabbdd mutant plants. The
photograph was taken 7 d after inoculation in planta

32. 16-April-18 PG seminar 32
Case study:3
objectives:
1. To disrupt the function of the recessive eIF4E (eukaryotic translation
initiation factor 4E) gene by Cas9/subgenomic RNA (sgRNA) technology.
2. To demonstrate the development of broad virus resistance in non
trangenic cucumber plants.

33. 16-April-18 PG seminar 33
Fig: 1 Gene editing of eIF4E mediated by CRISPR/Cas9 in transgenic cucumber plants

34. 16-April-18 PG seminar 34
Homozygous eif4e mutant plants exhibited immunity to Cucumber vein yellowing
virus (CVYV) infection.
Homozygous eif4e mutant plants exhibited resistance to Zucchini yellow mosaic
virus (ZYMV) infection

35. 16-April-18 PG seminar 35
Genome editing on the net
Organization Service provided
Addgene (http://www.addgene.org) Contains dozens of plasmids for creating ZFNs,
TALENs, or CRISPR
The Sheen and Gao laboratories Constructed a codon-optimized Cas9 for
Arabidopsis thaliana and rice (Oryza sativa) which
are available on request
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/cris
pr/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, 2016)

36. 16-April-18 36PG seminar

37. 16-April-18 37PG seminar