A seminar presentation on TALENs a tool most widely used in genome editing for building up desired traits in crop plants

1. 1972
2018
TO SINCE
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2. 2

3. 3
3
University of Agricultural and Horticultural Sciences, Shivamogga
College of Horticulture, Mudigere.
TALENs: A WIDELY APPLICABLE TECHNOLOGY FOR TARGETED
GENOME EDITING
BY:
MAHAMMED FAIZAN
MH2TAG0165
Dept. of genetics and plant breeding

4. SEMINAR OUTLINE
Genome editing
Timeline
Types of editing
Repairs mechanism
Technologies in genome editing
TALENs system
Construction of TALEN
Application
Experimental studies
Conclusion
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5. Fig. 01 Gene Editing Market Size, by Application, 2013- 2024 (USD Million)
(Global Market Insights, 2016)
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6. Genome Editing
‘Genome Editing’ or ‘Genome Editing with Engineered Nucleases’ (GEEN) Is
a tool Genetic engineering in which DNA is inserted, deleted or replaced in the
genome of a living organism using engineered nucleases or "molecular scissors."
6

7. 1989
HR- mediated
targeting
2000
Bacterial
Crispr-Cas
1998
ZFNs (Zinc Finger Nuclease)
1992
Cre- lox
1985
Human genome
editing
2010
N-C terminus discovery
2009
TALENs (Transcription Activator-
Like Effector Nuclease)
Genome editing
2011
RVD discovery
2013
Crispr-Cas
genome editing
2010
TALENs Genome
editing.
1989-95
Genome damage
repair
2011
Method of the year
2011
TALEN application in crop
plants
2009
TALE decoding
2007
TALE in bacteria
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8. Fig. 02 Types of genome editing
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9. DNA repair
DSBs can be repaired by one of two highly conserved cellular processes.
Nonhomologous end joining (NHEJ)
Homologous Recombination (HR)
Fig. 03 DNA repair 9

10. Early approaches of genome editing
• Random or non-targeted methods
*Ionizing radiation
*Chemical-induced mutagenesis
• Homologous recombination
• Transposans.
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11. Present tools for targeted genome editing
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Fig. 04 Meganucleases Fig. 05 Zinc-Finger Nucleases (ZFN)
Fig. 06 CRISPR Fig. 07 TALEN

12. • Transcription Activator-Like Effector
• Nucleases.
Are artificial restriction enzymes generated by fusing a TAL effector DNA
binding domain to a DNA cleavage domain.
TALENs
12
Fig. 07 TALEN

13. Highly conserved bacterial proteins
Xanthomonas sp.
Translocated into host cells by a Type III secretion system
Transcriptionally activate their corresponding host target genes
- virulence (ability to cause disease)
Dependent on the host genetic context
TALE
13

14. • Having FokI with customizable DNA binding domain
• TALEs are highly conserved repeat domains
• protein-DNA code relates to modular DNA binding TALE repeat domains to
individual bases in a target binding site.
• Generated by fusion
• Can target DNA sites up to 36-40 bp and can be engineered to bind desired
DNA sequence
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15. DNA binding code of TALEs domain
1. NI-A
2. HD-C
3. NG-T
4. NN-G
(Boch et al., 2012)
Fig. 08 Binding domain
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16. Fok I Domain
• Flavobacterium okeanokoites
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
A
T
C
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
G
T
A
G
G
5’
3’ 5’
3’
Nuclease
DNA-binding
Fig. 09 Fok I domain
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17. TALEN Design Guidelines
1. TALEN monomer binding sites
must be preceded by 5’-T
2. No T at position 1
3. No A at position 2
4. No G at position 3
5. Must not end with a G
6. Base composition more than 2
RVDS shouldn’t used.
Fig.10 Nucleotides and RVDs
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18. Software for TALEN design
• Used for design of TALEN and
TAL effectors for genome
editing.
• Guidelines reflect naturally
occurring TAL effectors .
• Binding sites
• Spacer lengths
 TALEN Hit™ Software.
TALE-NT
TALEN Access™
TALEN First™
 TALEN Sure KO™
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19. (TAL Effector Nucleotide Targeter 2.0)
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20. METHODS OF CONSTRUCTION OF TALENs
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Fig.11 Schematic of the strategies for the
assembly of TALE repeat arrays.
A: The REAL strategy based on hierarchical
ligations (Sander et al., 2011).
B: The Golden Gate cloning-based strategy
(Cermak et al., 2011).
C: The FLASH assembly method based on
solid-phase ligation (Reyon et al., 2012b).

21. Table 01. Examples of targeted gene modification using TALENs in plants
21
Arabidopsis ADH1 NHEJ Golden Gate Gene knockout Cermak et al., 2011
Tobacco EBE of Hax3 NHEJ Not available Gene knockout Mahfouz et al., 2011
Tobacco SurA, SurB NHEJ, HR Golden Gate Gene knockout, insertion
and replacement
Zhang et al., 2013
Rice EBE of
AvrXa7 and
PthXo3
NHEJ Golden Gate Gene knockout Li et al., 2012b
Rice OsDEP1 NHEJ Golden Gate Gene knockout, large
deletion, inversion
Shan et al., 2013
Rice OsBADH2 NHEJ Golden Gate Gene knockout Shan et al., 2013
Rice OsCKX2 NHEJ Golden Gate Gene knockout Shan et al., 2013
Rice OsSD1 NHEJ Golden Gate Gene knockout Shan et al., 2013
Brachypodium BdABA1 NHEJ Golden Gate Gene knockout Shan et al., 2013
Brachypodium BdHTA1 NHEJ Golden Gate Gene knockout Shan et al., 2013
Plant species Gene name Modification TALEN construction Mutation Reference type
platform

22. Main applications
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 Drug discovery.
 Quality traits.
 Gene function studies

23. Merits of targeted genome editing
• Gene modification
• Transgenic and Non-transgenic method
• Gene disruption in the therapeutic uses
• Efficient, easy in design, less toxic
• Reverse genetics approach
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24. Experimental Studies 24

25. Objective:-
To target the Os11N3 gene of
rice for genome editing using TALENs to
induce bacterial blight in rice.
Rice Bacterial Blight
(Li et al., 2012, USA)
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26. Causal organism:- Xanthomonas oryzae pv. oryzae (Xoo)
Gene:- Os11N3
TAL effectors:- AvrXa7 or PthXo3 effector
EBE:- effector-binding element
METHOLOGY :-
(Li et al., 2012, USA )
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27. Fig. 12 TALENs and their DNA targets in the promoter of
chromosomal Os11N3 gene. The two pairs of nucleases
(Pair 1 and 2) are fusions between the DNA cleavage
domain of FokI (FokI) and the native (AvrXa7) or
customized TAL effector (dTALE). Os11N3 promoter
contains an effector binding element (EBE) for AvrXa7, an
EBE for PthXo3 and the TATA box.
Fig. 13 Analysis of PCR
products derived from five
individual callus lines using
the Surveyor nuclease
cleavage assay) and
showing two out of five
calli (#1 and #2) with
biallelic mutations
Fig. 14 Schematic diagram of a two-gene expression cassette in
a single binary vector designed for Agrobacterium-mediated
rice transformation
(Li et al., 2012, USA )
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28. Fig. 15 Sequence of Os11N3 gene mutations in T1 plants
induced by the Pair 1 TALENs. Deletions and insertions are
indicated by dashes and red letters, respectively. TALEN-
binding sequences are underlined in the wild type (WT)
gene sequence. Numbers and letters designating each
individual mutant are indicated to the right side of the DNA
sequence.
Fig. 16 Additional haplotypes detected in T2 plants carrying
Os11N3 gene mutations produced with Pair 2 TALENs.
Deletions and insertions are indicated by dashes and red letters,
respectively. Numbers and letters designating each individual
mutant are indicated to the right side of the DNA sequence.
TALEN-binding sequences are underlined in wild type (wt).
(Li et al., 2012, USA )
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29. Fig. 17 Disease resistance in transgenic rice T1 plants. Lesion
lengths caused by infection with a pathogenic AvrXa7-
dependent Xoo strain were measured 14 days after inoculation
of 20 T1 mutant plants (1 – 20) generated from TALEN pair 2
and 2 wild type Kitake plants (21 – 22).. Leaf lesion lengths of
1~4 cm indicate disease resistance and lesion lengths of 10
~14 cm indicate disease susceptibility. Error bars indicate 1
SD.
Fig. 18 Severity of disease damage to wild type and
transgenic rice plants caused by AvrXa7-, PthXo3-, and
PthXo1-dependent Xoo strains. Lengths of lesions in wild
type plants (CK1), segregating T2 transgenic plants with
intact Os11N3 (CK2) and T2 transgenic plants homozygous
for Os11N3 promoter mutations of 6 bp (-6b), 9 bp (-9a), 15
bp (-15) and 4 bp (-4d) deletion
(Li et al., 2012, USA )
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30. Fig. 19 Gel images of PCR products obtained with the primer
sets of P1, P2 and P3 for hpt gene, TALEN genes and Os11N3
promoter, respectively. Labels below gel images represent: v,
binary vector DNA; ck+, a positive control of DNA from a
transgenic plant containing the T-DNA region depicted in (a)
and the Os11N3 gene; wt, DNA from a non transgenic, wild
type rice plant; number, individual T1 plants selected from
genetic crosses to lack the T-DNA region, but retain a
functional Os11N3 promoter region containing inactivated or
deleted AvrXa7 and PthXo3 EBE sites
Fig. 20 Resistance phenotype displayed by two T2
mutant plants compared with the disease susceptibility
phenotype of a non transgenic wt rice plant.
(Li et al., 2012, USA )
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31. (Lor et al.,2014, USA)
Aim:-
Targeted mutagenesis in tomato
(Solanum lycopersicum) of the negative
regulator of GA signaling PROCERA
gene using TALEN
Gene:- PROCERA (PRO) solyc11g011260.1
TAL effectors:-pTAL423/4 and pTAL425/6
promoter estrogen-inducible XVE
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32. Fig. 21 Location of TALEN target sites. Arrow depicts
PROCERA (Solyc11g011260.1) gene model. Underlined
sequences are the binding sites recognized by the TALEN
pairs
Fig. 23 Strategy for
regenerating pTAL425/6
TALEN-induced mutants.
A. Transgenic plants
expressing the pTAL425/6
TALEN are produced in the
first round of tissue culture in
the absence of β-estradiol to
avoid TALEN cytotoxicity.
B. pTAL425/6 T1 seeds are
collected from primary
transformants in A and
germinated for a second round
of tissue culture. Cotyledons
are excised and incubated
with β-estradiol to induce
TALEN expression, and
plants, designated as βE-
pTAL425/6 M0, are
regenerated and screened by
PCR to identify lines with
TALEN-induced mutations.
Fig. 22 DNA sequence alignment of TALEN-induced
mutations. Left and right TALEN-binding sequences are
underlined in the wild type. The sizes of deletions (-) and
insertions (+) are indicated to the right of the sequences.
The red dashes indicate the location of a 39-bp insertion in
pro_1. (Lor et al.,2014, USA)
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33. TALEN ID Repeat Number Spacer Length Repeat Variable Diresidues (RVDs) Target Sequence (5’Î 3’)
pTAL423 left 17 NI NG NI NI NI NN NG NN NI NI NN ATAAAGTGAAGTCGTCT
19 NG HD NN NG HD NG GATATGGCGGATGTTGCTC
pTAL424 right 26
NINNHD HD NI NG HD NG HD NI
AAAAACTTGAACAGCTTGAG
NI NN HD NG NN NG NG HD NI NI ATGGCT
NN NG NG NG NG NG
pTAL425 left 29
HD NG NN NI NG NI NG NN NN HD CTGATATGGCGGATGTTGC
NN NN NI NG NN NG NG NN HD TCAAAAACTT
NG HD NI NI NI NI NI HD NG NG
18 GAACAGCTTGAGATGGCT
pTAL426 right 16
HD HD NI NG HD NN NG NG NN ATGGGTACAACGATGG
NG NI HD HD HD NI NG
Table 02. TALEN RVD sequences and TALEN target sequences
(Lor et al.,2014, USA)
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34. Fig. 24
A. Three-week-old seedlings segregating for the proTALEN_2 allele.
Homozygous proTALEN_2 mutants are indicated by the asterisks.
B. Genotyping of the homozygous proTALEN_2 seedlings marked in A by
PCR amplification and digestion with Sm1l. C. indicates the no-template
control.
C. Comparison of 8-week-old wild-type (WT) and WT+GA3 plants with
homozygous proTALEN_2 plants.
D. Third youngest fully expanded leaves of the plants shown in C.
Fig. 25
A. Six-week-old seedling segregating for the
proTALEN_1allele
B. PCR–based genotyping confirmed the tall
seedlings in A are homozygous for proTALEN_1.
The no template control (C) is the negative
control. (Lor et al.,2014, USA)
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35. Aim:-
production high oleic acid peanut
varieties by using transcription activator-like
effecter nucleases (TALENs) mediated
targeted mutagenesis of Fatty Acid
Desaturase 2 (FAD2).
(Wen et al., 2018, China)
Gene:- ahFAD2B
TAL effectors:- L1R1 and L2R2 35

36. Fig. 27 TALENs targeting the AhFAD2 gene
A. Schematics of the AhFAD2 gene structures. The red
triangle and green triangle represent the two
potential TALEN target sites L1R1 and L2R2,
respectively.
B. Structure of a TALEN binding to its target gene
AhFAD2. The colored boxes denote the TAL effector
repeats
C. Structure of the last construct pCAMBIA1301-
TALENs-FAD2 for Agrobacterium-induced peanut
transformation
Fig. 26 Nucleotide sequences of TALEN-induced
mutations in peanut hairy roots. The TALEN-
binding sequences are shaded in gray in wild-type,
the TALEN-binding Sequences are in red letters, and
deletions are indicated by dashes
A
C
B
(Wen et al., 2018, China)
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37. TALEN ID
TALEN recognition sites
Total number of
hairy roots
Number of hairy
roots with mutations
Frequency (%)
L1R1
Left: CAA AAG AAG CCT CTT
216 18 8.33
Right: GAA TGG AGG GTT TGAA
L2R2 Left: GAA GCT CAA AAG AAGCC 105 13 12.38
Right: GAA TGG AGG GTT TGA
TALEN ID
TALEN recognition sites
Total number of
transformants in
T0
Genetic stable
mutants in T1
Frequency (%)
T1/T0
L1R1
Left: CAA AAG AAG CCT CTT 63 6 9.52
Right: GAA TGG AGG GTT TGAA
L2R2
Left: GAA GCT CAA AAG AAGCC 72 3 4.11
Right: GAA TGG AGG GTT TGA
Table 04. Numbers of FAD2 mutations induced by TALENs in the T0 and T1 generations
Table 03. TALEN activity assessment in peanut hairy-root assays
(Wen et al., 2018, China)
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38. Fig. 28 The fatty acid profile and agronomic characters of TALEN
induced FAD2 mutant lines.
A, B- The percentages of total fatty acid and protein content in
wild-type, L1R1 and L2R2 mutant lines in the T2 generation.
C, D- Relative contents of oleic acid and linoleic acid. Error bars
represent the standard deviation from the mean (n = 3). The
asterisks indicate a significant difference according to the t-test
(P < 0.05) compared with wild-type
Fig. 29 Phenotype of the wild-type and FAD2 mutant lines.
A. The photograph of the wild-type and FAD2 mutant lines in T1
generation.
B-D. The main agronomic traits in L1R1 and L2R2 mutant lines
compared with wild-type plant in the T2 generation. The scale bars
(Wen et al., 2018, China)
38

39. Aim:-
 To knockout OsBADH2 gene.
 To study Multiple gene knockout.
(Shan et al., 2015, China)
39

40. Gene:- fgr (OsBADH2)
TAL effectors:- T-OsBADH2b
Genes for multiplex knock out:- OsBADH2, OsCKX2 and OsDEP1
METHODS:-
(Shan et al., 2015, China)
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41. Fig. 30 TALEN-induced mutations in the OsBADH2 gene.
(a) The 2AP pathway in rice.
(b) (b) Schematic of OsBADH2 gene structure and TALEN binding
sites. The restriction enzyme site BgIII is highlighted in red.
(Shan et al., 2015, China)
Fig. 31 Segregation of the TALEN transgene in badh2 mutants. (a)
Schematic of the TALEN T-DNA construct showing the position of the
three pairs of PCR primers used to survey different region of the TALEN
transgene in the progeny of badh2 mutants.
A
B
Fig. 32 Sequencing of the TALEN-induced mutant alleles in each
of the triple-gene mutated rice plants. Deletions and insertions
are indicated by dashes and the ‘/’ symbol, respectively, and the
numbers on the right side show the sizes of the indels.
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42. T0 plant ID
T0
genotype
T0
mutation
type (bp)
Mutation segregation in T1 Transmissio
n ratio (%)*
TALEN-free
(%) †
Total Homo Hetero WT
badh2-1 Bb 3, 6, 12/+1 10 0 5 5 50 0
badh2-2 Bb 1 32 4 25 3 90.6
20.7
(6/29)
badh2-3 Bb 6 5 0 2 3 40 0
badh2-4 Bb 9 12 0 6 6 50 0
badh2-5 Bb 10 14 0 4 10 28.6 0
badh2-6 Bb 2, 6, 7 98 0 67 31 68.4 14.9 (10/67)
Table 05. TALEN-induced mutations in OsBADH2 and their transmission to the T1 generation
-n, nucleotide deletion of the indicated number; -n/+n, simultaneous nucleotide deletion and insertion of the indicated number at the same
site.
*Transmission ratio was calculated based on the number of plants carrying the mutations over the total number of plants tested.
†TALEN-free ratio was calculated based on the number of mutant plants not harbouring the T-DNA construct over the total number of plants
tested
(Shan et al., 2015, China)
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43. ‡Indicated that the segregation of the heterozygous lines (badh2-2-9) confirms to the Mendelian ratio (1 : 2 : 1) according to the v2 test (P >
0.5).
*Transmission ratio was calculated based on the number of plants carrying the mutations over the total number of plants tested.
†TALEN-free ratio was calculated based on the number of mutant plants not harbouring the T-DNA construct over the total number of
plants tested
T1 plant
ID
T1
genotype
T1
mutation
type (bp)
Mutation segregation in T2 Transmissi
on ratio
(%)*
TALEN-
free (%) †
Total Homo Hetero WT
badh2-2-6 bb 1 24 24 0 0 100 0
badh2-2-8 bb 1, 6, 10 24 24 0 0 100 0
badh2-2-15
bb
1 24 24 0 0 100
29.2 (7/24)
badh2-2-9 Bb 1 24 5 13 6 75‡ 0
Table 06. Genetic analysis of mutations in OsBADH2 and their transmission to the T2 generation
(Shan et al., 2015, China)
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44. Fig. 33 2AP contents of badh2-2 mutant grains as measured by GC-MS.
A-F:- Total ion chromatograms (TIC) of 2AP and TMP (as internal standard)
in the TALEN-induced badh2 mutant lines and control lines
G:- 2AP levels of the badh2 and control lines. Values are means SD of
three replications. A Student’s t-test was applied to generate P-
values.
(Shan et al., 2015, China)
44

45. No. of tested
plants
Mutations in OsBADH2 (%) Mutations in OsCKX2 (%) Mutations in OsDEP1 (%)
Total Homo Hetero Total Homo Hetero Total Homo Hetero
207 20 (9.7) 2 (1.0) 18 (8.7) 53 (25.6) 19 (9.2) 34 (16.4) 19 (9.2) 6 (2.9) 13 (6.3)
Single (%) Double (%) Triple (%)
No. of tested plants BADH2 CKX2 DEP1 BADH2/CKX2 BADH2/DEP1 CKX2/DEP1 BADH2/CKX2/DEP
1
207 6 (2.9) 30 (14.5) 2 (1.0) 8 (3.9) 2 (1.0) 11 (5.3) 4 (1.9)
Table 07. Multiple gene knockouts in rice using TALENs, showing the frequencies of mutations in each of the three
genes targeted
Table 08. Multiple gene knockouts in rice using TALENs, showing the frequencies of mutations
for all the combinations of single, double and triple genes targeted
(Shan et al., 2015, China)
45

46. (Clasen etal., 2015, USA)
AIM:-
To knockout vacuolar invertase gene (Vinv)
through TALEN to improve cold storage and
processing traits in potato.
Gene:- vacuolar invertase gene (VInv)
TAL effectors:- VInv_T1, VInv_T2 and VInv_T3
46

47. Fig. 34 Targeting the Solanum tuberosum cv Ranger Russet VInv gene with (TALENs).
(a) Compound sugar breakdown
(b) Schematic of the VInv gene. TALEN target sites are indicated with black, grey and
white triangles (VInv_T1, VInv_T2, VInv_T3, respectively).
(c) TALEN target sites within exon 1. Single nucleotide polymorphisms are indicated by
lowercase bold letters. Underlined letters indicate TALEN-binding sites.
A1, allele 1; A2(1), copy 1 of allele 2; A2(2) copy 2 of allele 2; A(3), allele 3.
Fig. 35 (a) Protoplasts from leaves on
3-week-old potato plants (Ranger
Russet) were isolated and
transformed with plasmids encoding
TALEN pairs. Following
transformation, exon 1 was amplified
by PCR and mutations were assessed
by 454 pyro-sequencing.
(Clasen et al., 2015, USA)
47
a
b
c

48. (a) Approach and timeline to
regenerate plants with mutations in
VInv. Protoplasts were transformed
with plasmids encoding the VInv_T2
transcription activator-like effector
nucleases pair and were cultured in
nonselective regeneration medium.
Following shoot and root formation,
potato plantlets were transferred to
soil
a
(b) Examples of plant lines carrying mutations in one or more of the
VInv alleles. WT, wild type.
b
(c) Images of potato chips after being
processed from tubers stored at 4 °C for 14
days.
c
(Clasen et al., 2015, USA)
Fig. 36 Recovery of potato lines carrying mutations within vacuolar invertase (VInv).
48

49. Fig. 37 Quality assessment of mutant potato lines.
(a) Analysis of sugar content within potato tubers stored at 4 °C for 14 days. Error bars represent standard deviation.
(b) Analysis of acrylamide content in potato chips that were processed from tubers that were stored at 4 °C for 14 days.
a b
(Clasen et al., 2015, USA)
49

50. INSTITUTES
• DBT, New delhi, India
• NHGRI, Bethesda, USA
• NCBI, bathesda, USA
• Cellectis Plant Science, New brighton, USA
• State Key Laboratory Of Plant Cell And Chromosome Engineering,
Beijing, China
50

51. DEMERITS
Require larger size of genome.
Dimerization.
Off targeting.
High cost.
Highly laborious.
Skilled persons.
51

52. Future directions in targeted genome editing…,
oAddressing Off target sites
o lead to unwanted mutation
oSequence information of crops.
oTargeting poly-genes
oCombining both binding domain and sequence specific (Type-II RE) enzymes,
oThe optimization of methods for efficiently delivering TALENs
oPlatforms for construction of TALENs.
52

53. GENETIC
ENGENEERING
DON’T MAKE
NEW GENES,
THEY
REARRANGE
EXISTING
ONE……. 53

54. 54
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