Content Introduction Types of genome editing Zinc Finger Nuclease Introduction Method TALEN Introduction Method for TALEN construction Applications Conclusions

1. 1

2. Recent Advances in Genome Editing
and its Applications
Presented by
Nandha Abhijeeta K.
2

3. Content
• Introduction
• Types of genome editing
• Zinc Finger Nuclease
Introduction
Method
Case study
• TALEN
Introduction
Method for TALEN construction
Case study
• Applications
• Conclusions 3

4. Genome Editing
• 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).
4

5. 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).
5

6. Cont…
• But sometime gene disruption
by siRNA can be variable or incomplete.
• Nucleases such as ZFNs or TALEN 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.
6

7. Types of Genome Editing
• There are currently four families of engineered
nucleases being used:
1. Mega-nuclease
2. Zinc finger nucleases (ZFN)
3. Transcription Activator-Like Effector-based
Nucleases (TALEN)
4. CRISPR/Cas system
7

8. 1. Meganucleases
Meganucleases were the first
class of sequence-specific
nucleases used in plants.
They continue to be
deployed to achieve complex
genome modifications.
An advantage of
meganucleases is their size.
Baltes and Voytas (2015)
8

9. They are among the smallest nucleases –
comprising only 165 amino acids (aa) –
making them amenable to most delivery
methods, including vectors with limited cargo
capacities, such as plant RNA viruses.
Relative to other sequence-specific nucleases,
however, mega-nucleases are challenging to
re- design for new target specificity.
Redesign is hindered by the non-modular
nature of the protein.
Baltes and Voytas (2015)
9

10. For example, within the LAGLIDADG family of
meganucleases, the amino acids responsible for
binding DNA overlap with those for DNA
cleavage; therefore, attempting to alter the
DNA-binding domain can affect the enzyme’s
catalytic activity.
As a result, the use of meganucleases in plants
has been limited to naturally occurring
meganucleases (e.g., I-SceI, I-CreI) or to
redesigned nucleases made by groups with
expertise in structure-based design.
Baltes and Voytas (2015)
10

11. 2. Zinc-finger nucleases
Like the meganucleases, zinc-finger nucleases are
relatively small (300 aa per monomer; 600 aa per
nuclease pair), making them amenable to most
delivery methods.
DNA targeting by zinc-finger nucleases is achieved
by arrays of zinc fingers, each of which typically
binds to a nucleotide triplet.
Baltes and Voytas (2015)
11

12. • Whereas redesigning the zinc-finger
DNA-binding domain is not as difficult as
for meganucleases, there are still
challenges in achieving new target
specificity.
• For example, a zinc finger that
recognizes GGG in one array may not
recognize this sequence when
positioned next to different zinc fingers.
Baltes and Voytas (2015)
12

13. Therefore, modular assembly of zinc fingers has
had limited success.
One of the more successful methods for
redirecting targeting involves screening libraries
of three zinc-finger variants to identify those
that best recognize and bind to their intended
target sequence.
More recently, modular methods for
constructing zinc-finger arrays have been
successful that use two-finger units to minimize
context effects.
Consequently, generating functional zinc-finger
nucleases is now achievable by most research
labs. Baltes and Voytas (2015)
13

14. 3. TALENs
• Transcription activator-like effector
nuclease (TALEN) technology
leverages artificial restriction
enzymes generated by fusing a TAL
effector DNA-binding domain to a
DNA cleavage domain.
• Transcription activator-like effectors
(TALEs) can be quickly engineered to
bind practically any desired DNA
sequence.
• By combining such an engineered
TALE with a DNA cleavage domain
(which cuts DNA strands), one can
engineer restriction enzymes that will
specifically cut any desired DNA
sequence.
Baltes and Voytas (2015)
14

15. • When these restriction enzymes are introduced into
cells, they can be used for gene editing, a technique
known as genome editing with engineered nucleases.
• TALENs are a recent addition to the arsenal of sequence-
specific nucleases, and they quickly became adopted for
plant genome engineering.
• This relatively large target site makes TALENs the most
specific of all the nucleases, and may contribute to
reduced toxicity compared to zinc-finger nucleases.
• TALENs are typically delivered to plant cells by direct
delivery of DNA to protoplasts, or by stable integration
of TALEN-encoding constructs into plant genomes.
Baltes and Voytas (2015)
15

16. 4. CRISPR/Cas
• The most recent addition to
the sequence-specific nuclease
family, Clustered Regularly
Interspaced Short Palindromic
Repeats which is commonly
known as CRISPR, is proving to
be the nuclease-of-choice for
plant genome engineering.
• Come into focus from studies
of how bacteria fight infection.
• A CRISPR array is composed of
a series of repeats interspaced
by spacer sequences acquired
from invading genomes.
Baltes and Voytas (2015)
16

17. This sequence is transcribed as crRNA which guides
CRISPR-associated (Cas) protein(s) to analogous
invading genomes introducing a DSB in the pathogenic
DNA, inhibiting integration and replication of the
pathogen.
Unlike the other three nuclease classes, which target
DNA through protein/ DNA interactions, CRISPR/Cas
uses a guide RNA molecule (gRNA) to direct an
endonuclease, Cas9, to a target DNA sequence.
As a result, redirecting CRISPR/Cas is extremely simple,
requiring only the cloning of a 20 nt sequence
(complementary to a target DNA sequence) with-in a
gRNA expression construct.
Baltes and Voytas (2015)
17

18. One limitation of the CRISPR/Cas system may be
off-target cleavage. Whereas 20 nucleotides are
used to direct Cas9 binding and cleavage, the
system tolerates mismatches.
To reduce the likelihood of off-target cleavage,
alternative CRISPR/Cas reagents have been
developed, including paired Cas9 nickases, fusion of
catalytically-dead Cas9 to FokI, and shortening of
the gRNA targeting sequence.
Baltes and Voytas (2015)
18

19. 19

20. Name Components
Mechanism of
action
Specificity/off-
target effect
Possibility to
rapidly generate
large-scale libraries
Meganucleases
Endonuclease with a
large recognition for
DNA (12-40 base
pairs)
Induces double-
strand breaks in
target DNA
Highly specific
No-limited target
sequence
specificity available
Zinc finger
nucleases
(ZFNs)
Fok1 restriction
nuclease fused to
multiple zinc finger
peptides; each
targeting 3 bp of
genomic sequence
Induces double-
strand breaks in
target DNA
Can have off-
target effect
No-requires
customization of
protein
components for
each gene
Transcription
ativator-like
effector
nucleases
(TALENs)
Non-specific DNA-
binding domain
specific for a
genomic locus
Induces double-
strand breaks in
target DNA
Highly specific
Feasible, but
technically
challenging
CRISPR/Cas9
20nt crRNA fused to
tracrRNA and Cas9
endonuclease
Induces double-
strand breaks in
target DNA (wt
Cas9) or single-
strands DNA nics
(Cas9 nickase)
Some off-target
effects that can be
minimized by
selection of
unique crRNA
sequences
Yes- requires simple
adapter cloning of
20nt Oligos
targeting each gene
into a plasmid
Heintze et al., (2013) 20

21. Genome modifications achieved in plants
using sequence-specific nucleases
21

22. Type of DNA
modification
Nucleases Delivery method (s) Plants Target(s)
Trait staking
Meganuclease Bombardment Cotton
Intergenic
sequence
ZFN Bombardment Zea mays Transgene
Gene Knockout
Meganuclease
Stable integration ;
RNA Virus
Zea mays
Intergenic
sequence
Meganuclease
Stable integration ;
Agrobacterium T-DNA
(transient)
Zea mays MS26
ZFN Stable integration
Arabidopsis
thaliana
ADH1, TT4
ZFN Stable integration Glycine max
DCL1a/b,
HEN1a
ZFN Stable integration
Arabidopsis
thaliana
Transgene
TALEN Protoplast Arabidopsis
thaliana;
Tobacco
AtTT4, AtADH,
NbSurB
TALEN Bombartment Triticum
aestivum
MLO
CRISPR/Cas Stable integration Arabidopsis ADH1; TT4
Baltes and Voytas (2015)
22

23. Type of DNA
modification
Nucleases Delivery method (s) Plants Target(s)
Large deletion
ZFN Stable integration Tobacco Transgene
ZFN Stable integration Arabidopsis
thaliana
RPP4 gene cluster
CRISPR/Cas Protoplasts
Arabidopsis
thaliana
PDS3
CRISPR/Cas
Protoplasts; stable
integration
Oryza sativa
Labdane-related
diterpenoid gene
clusters on Chr 2,
4 and 6
Gene
replacement
Meganucleases
Agrobacterium T-DNA
(transient)
Tobacco Transgene
Meganucleases Agrobacterium T-DNA
(transient)
Tobacco Transgene
ZFN
Agrobacterium T-DNA
(transient; donor only)
Arabidopsis
thaliana
PPO
ZFN Whiskers Zea mays Transgene
TALEN Protoplast Tobacco SurA/B
CRISPR/Cas Nicotiana 23

24. Zinc Finger Nuclease
24

25. Structure of Zinc Finger Nuclease
ZNF is a protein motif which
contain a bound Zn ion and a
protein “finger”.
It was first discovered in
Xenopus laevis (african
clawed frog) as the DNA
binding domain of
transcription factor.
It is classified in different
ways
1) Numbers of cysteines and
histidines (typical zinc
finger motifs are composed
of two cycteines followed
by two histidines)
2) Structure
25

26. Classification of Zn fingers
• Zn fingers can be structurally divided into 8
classes.
• All members have different binding
properties, within class as well as between
class.
• They bind to DNA, protein and small
molecules.
• Many are coordinated by Zn ions, but not all.
26

27. Fold group Representative structure Ligand placement
C2H2
Two ligands from a knuckle and two
more from the C terminus of a helix
Gag knuckle
Two ligands from a knuckle and two
from a short helix or loop
Treble clef
Two ligands from a knuckle and two
more from the N terminus of a helix
Zinc ribbon Two ligands each from two knuckles
Zn2/Cys6
Two ligands from N terminus of a helix
and two more from a loop
TAZ2 domain like
Two ligands, each from the termini of
two helices
Zinc binding loops Four ligands from a loop
Metallothionein
Cysteine rich metal binding loop
Krishna et al. (2003) 27

28. Zn fingers bind DNA in a sequence specific
manner, usually specific to a 3 nucleotide
codon.
The small motif can easily be incorporated
as units in a larger protein. These can be
engineered to bind different sequence of
varying length.
It also can be used to target other protein
functional domains to a specific DNA
sequence.
28

29. Mechanism of ZFN
29

30. • A series of Zn finger motifs bound to a DNA
nuclease in which, the Zn fingers bind to a
specific DNA sequence while the nuclease
induces a double strand break at the site.
30

31. 31

32. Mode of action of ZFN
32

33. 33

34. Advantages of ZNF
• High fidelity
• Site specific insertion
• Point mutation-can induce sequence
change in native genes
• Very few extraneous DNA sequences in
the final product.
34

35. Potential concern of ZNF
• Off target cleavage
 Sequence similarity
“Homodimer”
sequence match
Could lead to
unintended
mutation
35

36. TALEN
36

37. Transcription activator-like effector nuclease
• TALEN stands for Transcription activator-like
effector nucleases.
• TALEN is derived from molecules called as TALE,
to which a endonuclease enzyme is fused.
• TALE is produced by a plant pathogen-
Xanthomonas via a type III secretion system.
• Xanthomonas is a member of Proteobacteria
known to infect plants. Upon a successful
infection the TALE binds host genome and
modulates its expression of variety of proteins.
37

38. TALE
38

39. Structure of TALEN
• Structurally TALE consists of 18 repeats of 34
amino acids.
• The repeat varies at amino acids 12 and 13. These
variation are called as RVD (Repeat Variable
Diresidue).
• Different RVDs associate preferentially with
different nucleotides, with the four most
common RVDs (HD, NG, NI, and NN) accounting
for each of the four nucleotides (C, T, A, and G,
respectively).
39

40. Cont…
• RVD is highly variable and show a strong
correlation with specific nucleotide recognition.
• This relationship between amino acid sequence
and DNA recognition has allowed for the
engineering of specific DNA-binding domains.
• Slight changes in the RVD and the incorporation of
"nonconventional" RVD sequences can improve
targeting specificity.
40

41. By fusing the TALE sequence to a Fok1 nulcease,
the synthetic compound is now called a TALEN.
However, Fok1 works only in a dimerized form
and hence TALENs are always designed as pairs
binding opposing strands of the DNA to allow
dimerization of FokI in a spacer region that is
bridging the two TALE binding sites.
The treatment of genome with TALEN lead to
specific DNA binding and subsequent DSB
(Double strand break) which the genome repairs
through a NHEJ or HDR.
41

42. Methods for Construction of TALEN
TALEN
Golden gate
cloning base
assembly
GG
(golden
gate)
GG-PCR
Sequential
assembly
UA
(unit
assembly)
REAL/REAL
-Fast
(restriction
enzyme
and
ligation
High-throughput
solid-phase assembly
FLASH
Fast
Ligation-
based
automatable
solid phase
high-
throughput
ICA
Iterative
capped
assembly
http://eendb.zfgenetics.org/util-construc-t.php
42

43. Creating TAL Effectors and TALENs via Golden Gate
assembly
Cermak et al. (2011) 43

44. Construct Assembly Timeline
Cermak et al. (2011)Minnesota
44

45. 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
45

46. Advantages of using TALEN
High specificity to the target
Successfully used in
combination with the catalytic
domain of many enzymes.
No off target cleavage.
Software developed is free
online tool.
46

47. Disadvantages
Sensitive to DNA methylation of the target
region.
Time consuming and more elaborate
synthesis.
Complication in delivery because of it’s large
size.
47

48. ZFN vs. TALEN
Characteristic ZFN TALEN
Origin of DNA recognition
unit
DNA binding motif within
transcription factors
Structural repeats within
the secreted
transcriptional regulatory
proteins from plant
pathogenic bacteria
Size of DNA recognition
unit
Around 30 amino acids
per unit targeting a 3bp
sequence
Around 34 amino acids
per unit targeting single
base
Modular assembly of
DNA recognition units
Possible, but limited by
the interaction between
neighboring units
Possible
Presence of repeated
units
As zinc finger domains in
tandem
As TALE DNA binding
domains in tandem
48

49. Applications of Genome Editing
• The rapid development of customized ZFNs has
already substantially expanded the scope of genetic
research that can be performed in a broad range of
different organisms and cell types.
• The high efficiencies of alterations observed have
already inspired efforts to use ZFNs as a potential
therapeutic approach for genetic-based diseases.
• The relative simplicity with which TALENs can be
engineered will further spur efforts to explore the
research and therapeutic applications of customized
nuclease technology as well as in disease control.
49

50. Conclusions
Genome editing tools provide new strategies for genetic
manipulation in plants and are likely to assist in engineering desired
plant traits by modifying endogenous genes.
Genome editing technology will have a major impact in applied
crop improvement and commercial product development .
Both techniques, ZFN and TALEN, will no doubt be revolutionized by
virtue of being able to make targeted DNA sequence modifications
rather than random changes.
In gene modification, these targetable nucleases have potential
applications to become alternatives to standard breeding methods
to identify novel traits in economically important plants and more
valuable in biotechnology as modifying specific site rather than
whole gene.
50